Solid-Phase Synthesis of Compound Libraries Around the 1,5-Benzodiazocine-2,6-Dione Privileged Structure-Like Scaffold

SOLID-PHASE SYNTHESIS OF COMPOUND LIBRARIES AROUND THE 1,5-BENZODIAZOCINE-2,6-DIONE PRIVILEGED STRUCTURE-LIKE SCAFFOLD

Heba El-Metwally

Supervisor: Prof. Dr. Johan Van der Eycken

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Science: Chemistry

Academic year: 2016 – 2017

Members of the examination committee

Chair: Prof. Dr. Frederic Lynen (UGent)

Other members: Prof. Dr. Erik Van der Eycken (KU-Leuven) Prof. Dr. Kourosch Abbaspour Tehrani (UAntwerpen) Prof. Dr. Johan Winne (UGent) Dr. Jurgen Caroen (UGent) Prof. Dr. Johan Van der Eycken (UGent, promotor)

Acknowledgement

The journey started at once the decision about my Doctorate preparation was made, and after an intensive period of four years, today is the day: writing this note of thanks is the finishing touch of my thesis. It has been a period of intense learning and development for me, not only in the scientific arena, but also on a personal level. Thanks God for the uncountable blessings.

While a completed dissertation bears the single name of the student, the process that leads to its completion is always accomplished in combination with the dedicated work and support of other people. I wish to acknowledge my appreciation to the one who firstly believed in my potential, late Prof. Mohamed Abbas Metwally (God rest his soul), my first Organic Chemistry professor and true friend. You had the dream of your little daughter’s success, and I wish you were here to see it. You always supported, cared and gave me your time freely.

I would like to thank my promoter Prof. Johan Van der Eycken for giving me the opportunity to join this project through his great laboratory and be a part of your team. Thanks also go to Dr. Jurgen Caroen for sharing his knowledge and experience of solid phase synthesis, guiding me on ways of solving problems and keeping me inspired. Thanks to Eng. Jan Goeman. This project would have been extremely difficult without your LCMS analysis and other analyses.

I also would like to thank my colleagues for the pleasant and co-operative environment they provided; Karel-Simon, Timoty, Pieter, Fréderique, Sam, Bo, Dries, Nick, Wim and Mohamed; also the LOBOS girls Katrien, Xenia, Divya, Visnja, and Vineeta. I really enjoyed every minute with you, our trips, talks and food we shared.

Moreover, lots of thanks to Tom P., Tom D., Veerle, Paul and Karine for their administrative and important supporting services.

I wish to express my unqualified thanks and the special debt to my beloved husband, Dr. Mohamed Hammouda El-Mettwaly. You were always there in the ups and down of this research and life. I could never have accomplished this dissertation without your love, support, patience and understanding.

I was also blessed by the beautiful family, whose presence made my life happier. My mother and father Dr. Eman Sadek, late Prof. Mohamed Metwally; were a secret source of happiness and energy. Their unconditional love and sacrifices are behind my success. Brothers Dr. Ahmed Abbas, Eng. Amr Abbas and Vet. Ehab Abbas, your continuous motivation and support kept me going. Thanks to my sisters in law Sana and Mirna, for your encouragement and support. Kadria and Fawzia my lovely grandmothers, who knelt and prayed silently.

I am very grateful to friends I met in Belgium. Your presence in my life was such a second family outside home and an invaluable source of friendly support.

Dozens of thanks to Eng. Amr Metwally, Eng. Muhannad Ajjan and Eng. Sana Ajjan; you were always the analysts and solvers of my personal computer bugs.

Last but certainly not least, I am very grateful to the Egyptian ministry of Higher Education for funding me for 4 spent years at Gent University, Belgium.

Heba Metwally May 2017

Content

Part 1. Introduction

I. Medicinal Chemistry 1 1. History of medicinal chemistry from herbalism to drug design 1 2. Privileged structures 5 II. Combinatorial Chemistry 12 1. Definition & principles 12 2. History of combinatorial Chemistry 12 3. Solid phase synthesis as a tool for combinatorial chemistry 16 1. Solid supports 17 2. Linkers 19 4. Analysis of solid phase intermediates 20 1. On bead analysis methods 20 2. UV quantification method of resin cleavable chromophore groups 21 III. Literature Review on Benzodiazocine Derivatives 27 1. Introduction 27 2. 1,2-Benzodiazocines 30 3. 1,3-Benzodiazocines 31 4. 1,4-Benzodiazocines 32 5. 1,5-Benzodiazocines 37 6. 1,6-Benzodiazocines 43 7. 2,3-Benzodiazocines 49 8. 2,4-Benzodiazocines 50 9. 2,5-Benzodiazocines 51 10. 3,4-Benzodiazocines 54 Part 2. Results and Disscusion

IV. Aim and strategy 58 1. General aim 58 2. Synthesis strategies 58

3. Specific aim of this work 66 V. Solid phase synthesis of a model 1,5-benzodiazocine-2,6-dione 70 1. Selection of the solid support 70 2. Coupling of Fmoc-β-amino acid on Wang resin and Fmoc removal 70 3. Mitsunobu-Fukuyama Alkylation Sequence 71 4. Coupling of Fmoc-anthranilic acid derivatives 73 5. Fmoc removal and cleavage from resin 75 6. Cyclization in solution 76 VI. Synthesis of 3-substituted-1,5-benzodiazocine-2,6-diones 84 1. Retrosynthesis 84 2. Building block syntheses 85 3. Solid phase synthesis of 3-substituted-1,5-benzodiazocine-2,6-diones 102 4. Conclusion 111 VII. Synthesis of 4-substituted-1,5-benzodiazocine-2,6-diones 117 1. Retrosynthesis 117 2. Building block syntheses 118 3. Solid phase synthesis of 4-substituted-1,5-benzodiazocine-2,6-diones 119 4. Conclusion 127 VIII. Synthesis of 3,3-disubstituted-1,5-benzodiazocine-2,6-diones 130 1. Retrosynthesis 130 2. Building block synthesis 131 3. Solid phase synthesis of 3,3-disubstituted-1,5-benzodiazocine-2,6-diones 135 4. Conclusion 140 IX. Conformational analysis 142 1. Introduction 142 2. Conformational behavior of eight-membered benzo(bis)lactam-relevant literature 144 3. Conformational analysis of 1,5-benzodiazocine-2,6-diones 150 4. Conclusion 163

X. General conclusion and future perspectives 166 XI. English summary 170 XII. Dutch summary-Nederlandse samenvatting 184

Part 3. Experimental

XIII.1 Instrumentation and methods 188

1. Solvents and reagents 188 2. Purification 188 3. Characterization 188 4. Equipment 191 XIII.2 Synthesis of N-Fmoc-protected anthranilic acid derivatives 192

XIII.3 Synthesis of N-Fmoc-3-amino-2-alkylpropionic acids 198

1. Synthesis of methyl 3-aryl-2-cyanoacrylates 198 2. Synthesis of 3-substituted methyl 2-cyanopropionates 202 3. Synthesis of methyl 3-(tert-butoxycarbonyl)amino-2-alkylpropionates 207 4. Synthesis of 3-(tert-butoxycarbonyl)amino-2-alkylpropionic acids 212 5. Synthesis of HCl salts of 3-amino-2-alkylpropionic acids 217 6. Synthesis of 3-(9-Fluorenylmethyloxycarbonyl)amino-2-alkylpropionic acids 217 XIII.4 Synthesis of 3-substituted-1,5-benzodiazocine-2,6-diones 222

1. β2-Amino acid coupling on Wang resin 222 2. Nosyl coupling 223 3. Mitsunobu alkylation 224 4. Nosyl removal 225 5. Coupling of Fmoc-anthranilic acids 226 6. Fmoc removal 227 7. Cleavage from the resin 227 8. Cyclization in solution 238 XIII.5 Synthesis of N-Fmoc-3-amino-3-alkylpropionic acids 251

XIII.6 Synthesis of N-Cbz-4-aminobutan-1-ol 254

XIII.7 Synthesis of 4-substituted-1,5-benzodiazocine-2,6-diones 255

1. β3-Amino acid coupling on Wang resin 255 2. Nosyl coupling 256

3. Mitsunobu alkylation 257 4. Nosyl removal 258 5. Coupling of Fmoc-anthranilic acids 259 6. Fmoc removal 259 7. Cleavage from the resin 260 8. Cyclization in solution 270 XIII.8 Synthesis of N-Fmoc-3-amino-2,2-dialkylpropionic acid 281

1. Synthesis of methyl-2-cyano-2,5-(dimethyl)methylpentanoate 281 2. Synthesis of methyl-2-cyano-2-(naphth-2-yl)methyl)propionate 282 3. Synthesis of methyl 2-methyl-3-(naphth-2-ylmethyl)-3-(tert- butoxycarbonyl)aminopropionate 283 4. Synthesis of 2-methyl-3-(naphth-2-ylmethyl)-2-(tert-butoxycarbonyl)aminopropionic acid 285 5. Synthesis of HCl salt of 2-methyl-2-(Naphth-2-ylmethyl)aminopropionic acid 286 6. Synthesis of 3-(9-fluorenylmethyloxycarbonyl)amino-2-methyl-2-(naphth-2- ylmethyl)propionic acid 286 XIII.9 Synthesis of 3,3-disubstituted-1,5-benzodiazocine-2,6-diones 288

1. β2,2-Amino acid coupling on Wang resin 288 2. Nosyl coupling 289 3. Mitsunobu-Fukuyama alkylation 289 4. Nosyl removal 290 5. Coupling of Fmoc-anthranilic acid 290 6. Fmoc removal 291 7. Cleavage from the resin 291 8. Cyclization in solution 292

PART 1: INTRODUCTION

Part 1: Introduction

I. MEDICINAL CHEMISTRY 1. HISTORY OF MEDICINAL CHEMISTRY: FROM HERBALISM TO DRUG DESIGN. I.1.1 Era of herbalism Human health is a primary concern to mankind. As such, throughout history and around the world man has been actively searching among available natural resources to increase chances of survival and has discovered by trial and error, an arsenal of roots, berries , barks and leaves that could be used to mitigate symptoms of illness.

Perhaps as early as 4,000 BC1,2 in India, Ayurveda medicine has used numerous herbs such as turmeric roots, as evidenced by Sanskrit writings like the Rig Veda. In old Iraq by 3,000 BC in Mesopotamia, the Sumerians left clay tablets with hundreds of medicinal plants such as opium and myrrh3. By 2,000 BC, Chinese started prescribing herbal medicines4 and seeds, likely used for herbalism, have also been found in archaeological sites of the Bronze Age5. By 1550 BC, Egyptian texts (papyrus) are of particular interest due to the language translation that join texts from this region 6. The translated papyrus consist of lists of diseases, their treatments and has information on more than 850 plant medicines, including juniper, cannabis, garlic, castor , aloe, bean7,8. For instance, depicted in figure I.1, the Ebers Papyrus contains a prescription for Cannabis sativa (marijuana) applied topically for inflammation. Figure I.1: The Ebers Papyrus

Published around 400 BC, the Hippocratic Corpus book is a combination of around 60 early Ancient Greek medical works strongly related with the physician Hippocrates and his teachings, describing popular and predominant treatments of the early ancient Greek period9. Galen, a Greek physician practicing in Rome, was obviously productive in his attempt to write down his knowledge about medicine and notes for the follow up of the patients, resulting in many texts regarding herbs and their properties10. During the Middle Ages (400-1500), there were three major sources of information. Firstly, the Canon of Medicine from the Arabian school is known for the discovery of infectious diseases, the study of physiology and sexually transmitted diseases. The Canon includes a description of some 760 medicinal plants and the medicine that could be derived from them11. Secondly, Anglo-Saxon Leechcraft series consists of three books (known as Bald’s

Part 1: Introduction

Leechbook I, II and III), presenting a collection of treatment organized by ailment. The books are a collation of both Mediterranean and English medical practice12. Thirdly, Salerno was a famous school in Italy, concentrating on health and medicine. A student of the school was Constantine, credited with getting Arab medicine to Europe12.

I.1.2 Introduction of modern medical science at the molecular level

From this overview, we can therefore say that the history of medicine is closely tied with the history of herbalism from prehistoric times up to the introduction of the germ theory of diseases (Italian entomologist: Agostino Bassi between 1808 and 181313) and the birth of organic synthesis (Wöhler, 1828). In contrast, the medicinal chemistry from the 19th century onward is based on evidence assembly using scientific methods, and as a result synthetic drugs have largely substituted herbal treatments in epochal health care. In the nineteenth century, with the development of organic chemistry and chemical analysis the door towards the isolation and characterization of numerous active plant principles was opened. Soon, chemists tried to manipulate these compounds chemically to improve their activity and to reduce their side effects. Examples of compounds isolated from nature and synthesized include: Urea (described in 1727 by Herman Boerhaave14, anf first artificially organic compound ever synthesized in 1828 by Wöhler), and Morphine I.1 (isolation by Friedrich Sertürner15, 16 in 1805 - synthetic analogue Heroin I.2 was first synthesized in 1874 by C.R. Alder Wright).Furthermore, Quinine I.3 is used to prevent and treat malaria and babesiosis18, 19 (isolation in 1820 by Pierre Joseph and Joseph Caventou - first total synthesis in 1943 by RB Woodward and WE Von Doering17). Salicin I.4 is the active extract of willow bark, was isolated and named by the German chemist Johann Buchner in 182820, and is often used to treat pain, fever and inflammation21 (isolation 1828 - synthetic analogue aspirin 1853). Acetylsalicylic acid (Aspirin I.5) was first synthesized by Frederic Gerhardt in 1853. The discovery of Penicillin I.6 in 1928 by Fleming was a great landmark in the history of drug research22. His research was completed up by Florey and Chain in 1938 who could eventually industrialize the process of fermenting and extracting penicillin23. For their work on penicillin they were rewarded the Nobel Prize in Medicine in 1945. The first orally active semisynthetic steroidal estrogen is 17α-ethynylestradiol I.7 (isolation in 1929 by Adolf Butenandt and Edward Doisy - semi synthesized in 1938 by Herloff Inhoffen and Walter Hohlweg), and was used for diseases such as amenorrhea, breast cancer, hypogonadism, menopausal disorders, postpartum

Part 1: Introduction breast engorgement, and prostatic cancer. This synthetic derivative is 15-25% more active than the natural estradiol when administrated orally25. The isolation and determination of estrogen structure24 accelerated the hormonal drug research.

HO RO OMe HO H N O O O H OH HO O OH N H H OH N OH OCOCH3 RO I.1 morphine, R= H I.3 quinine I.4 D-(-)-salicin I.5 Aspirin I.2 heroin, R = COCH3 R2 R3 R OH 1 O N R H N H N S H O O N H H O HO COOH HO O

I.6 penicillin I.7 17-ethynylestradiol I.8 Camptothecin R1=R2=R3=H I.9 Topotecan R1=OH, R2=CH2-NMe2, R3=H O

I.10 Irinotecan R1= O N , R2=H, R3=Et O O OH O N

O O NH O HO O HO O O OH O

I.11 Paclitaxel

Figure I.2: Examples of identified compounds isolated from natural sources and synthesized derivatives thereof.

As a cytotoxic quinoline alkaloid isolated from the “Happy tree”, Camptothecin (CPT) I.8 (isolation in 1966 by M. E. Wall and M. C. Wani- synthesized 1992) inhibits the DNA enzyme topoisomerase. Because of low solubility and high reverse drug reactions, synthetic and medicinal chemists have developed numerous syntheses for Camptothecin26, 27 and its derivatives.

Part 1: Introduction

Topotecan I.9 and Irinotecan I.1029 are two CPT analogues which have been approved and used in cancer chemotherapy28 today. Paclitaxel I.11 is used to treat ovarian, breast, lung, pancreatic and other cancers (isolation in 1967 by M. E. Wall and M. C. Wani30, 31 - synthesized in 1994 by Robert Holten32, 33), marketed in 1989 by Bristol-Myers Squibb as taxol®.

I.1.3 Advancements towards rational drug design

Predominantly referred to as rational drug design is the contrived process of finding new medications based on the knowledge of biological targets34. The drug is most commonly a small organic molecule that stimulate or inhibits the function of a biomolecule such as protein, which in turn results in a therapeutic benefit to the patient. There are two major types of drug design35: ligand-based or structure-based.

I.1.3.1 Ligand-based

Based on knowledge of other molecules (ligands) able to bind to the biological target of interest, a pharmacophore model can be established that includes the necessary structural characteristics in order to bind to the target36. In addition a quantitative structure activity relationship (QSAR), in which a correlation between calculated properties of molecules and their experimentally determined biological activity, may be derived37.

I.1.3.2 Structure-based

Based on information of the three-dimensional structure of the biological target obtained through methods such as NMR spectroscopy or X-ray crystallography38. Computational medicinal chemists can identify potential new drug candidates by evaluating the binding affinity to the biological target39 in silico. The first unequivocal example of the application of structure- based drug design leading to an approved drug is the carbonic anhydrase inhibitor dorzolamide I.12, (figure I.3) which was approved in 199540.

Part 1: Introduction

O O S S O S NH2 O HN

I.12

Figure I.3: Structure of dorzolamide I.12.

I.2. PRIVILEGED STRUCTURES

I.2.1 Definition and characterization

The retrospective analysis of the chemical structures of the many drugs used in medicine led medicinal chemists to identify molecular motifs that are more frequently associated with high biological activity than other structures. Such molecular motifs were coined privileged structures by Evans et al., 41 to represent substructures that confer activity to two or more different receptors. Though he was originally referring to the benzodiazepine nucleus I.13(41, 42), which is thought to be privileged because of its ability to structurally mimic beta peptide turns43, work over the past several decades has revealed that there are additional such scaffolds. Although still unclear why, such structures appear to have the intrinsic property of binding with biochemical receptors44.

Among the most popular privileged structures, historical representatives are arylethylamines (including tryptamines I.14), diphenylmethane derivatives I.15, tricyclic psychotropics I.16 and sulfonamides I.14. Dihydropyridines I.1845, biphenyl I.19 and pyridazines I.2044 are other examples of such recurring motifs as privileged structure such as benzopyrans I.21, isoxazoles I.22, and monosaccharides I.23 46 (figure I.4).

Part 1: Introduction

R2 N R1

H O NH O O N 2 N S R3 R1 N N N H R2 I.13 I.14 I.15 I.16 I.17 HO

HO H N O O N N N O HO OH OH I.18 I.19 I.20 I.21 I.22 I.23

Figure I.3: Some examples of privileged structures.

Based on the chemical diversity of privileged structures and their intrinsic binding properties, in addition to favorable physiochemical properties and 3D-shape, it was expected that their use as scaffolds in combinatorial libraries would lead to the discovery of multiple active molecules for a variety of therapeutic targets. Combinatorial libraries based on the 1,4-benzodiazepin-2-one privileged structure were first synthesized by Ellman et al.47. From a 1680 member library, 3 compounds were identified by affinity for three different receptors. Nicolaou and co-workers, used the naturally occurring benzopyran scaffold for finding highly active agonists for the farnesoid X receptor48 and Schultz and Legraverend used the purine scaffold49.

Besides the use of already known privileged structures for finding new biological activity, there is also interest in the identification of new privileged structures. As a consequence more computational techniques are deployed via a slightly more reasoned approach50. Studies have shown that a remarkable proportion of privileged substructures suitable for combinatorial applications meets the following structural features: 1) the central motif is a bicyclic or tricyclic basic skeleton, as it provides a degree of molecular rigidity upon binding, resulting in less entropy loss than to "open-chain structures,"; 2) the molecular weight should be low enough to permit the introduction of the necessary extra substituents without causing violation of Lipinski’s Rule of five51 (i.e. MWscaffold <300 Da); 3) there is a relatively simple synthetic access to the basic

Part 1: Introduction skeleton, preferentially via automatable methods; 4) there should be a possibility to introduce a wide variety of substituents through different attachment points to explore the peripheral space.

I.2.2 Benzodiazepines as a privileged structure and inspiration for new skeletons

The classic example among the so-called privileged structures is the 1,4-benzodiazepine skeleton, where the first benzodiazepines (Librium® I.24, Valium® I.2552, figure I.5) were found to have significant activity in the central nervous system. The success of the structural privileged 1,4- benzodiazepine skeleton induced a rush of syntheses of new-closely related bioisosteric analogues. For instance, the rearrangement or substitution of the atoms in the diazepine moiety led to all kinds of new pharmaceutically interesting compounds (figure I.5). Moving one or both of the nitrogen atoms, for example, led to biologically active 1,5- and 2,3-benzodiazepines (I.26 and I.27)53,54, while the further introduction of heteroatoms led to 1,3,4-benzotriazepines55 for example (I.28).

Several of the derived classes of structures (for example, 1,5-benzodiazepines I.26 and 1,4- benzodiazepine-2,5-diones I.29)56 have a broad range of diverse biological activity and may be nowadays themselves considered to be privileged structures.

Part 1: Introduction

HOOC

NH O O O O N N N NH N Cl N Cl N O N O O

Chlordiazepoxide Diazepam Interleukin-1b converting Librium, 1960 Valium, 1963 enzyme (ICE) inhibitor, 2000 I.24 I.25 I.26

O O H O O O N O HO N N N O N N MeO N O HO F

H2N AMPA- receptor antagonist Peripheral benzodiazepine receptor (RK-1441B) antibiotic 1999 antagonist, 1984 1990 I.29 I.27 I. 28 Figure I.5: Small structural changes in the classical 1,4-benzodiazepine skeleton give rise to new classes of biologically active compounds.

In addition to the above changes in the benzodiazepine skeleton by atomic substitutions, potentially interesting analogues may also be discovered by modification of the ring size of the skeleton, e.g. by adding one more carbon atom to have eight-membered ring analogs, the so-called benzodiazocines which are the subject of this PhD-thesis. More data about the synthesis and biological activity of known derivatives of different skeletons of benzodiazocines can be found in chapter III.

Part 1: Introduction

References

1 Wynn, S.; Fougère, B. (2006). Veterinary Herbal Medicine. Elsevier Health Sciences press. 2 Aggarwal, B. B.; Sundaram, C.; Malani, N.; Ichikawa, H. Adv Exp Med Biol 2007, 595, 1-75. 3 Sumner; Judith (2000). The Natural History of Medicinal Plants. (p 17) Timber Press. 4 Hood; Lucy. (2006) Conceptual Bases of Professional Nursing. (p 35). Philadelphia: Lippincott Williams & Wilkins. 5 Hong; Francis McGill Journal of Medicine 2004, 8(1), 79-84. 6 Nunn, J. F. (1996). Ancient Egyptian Medicine. (p 6) University of Oklahoma Press. 7 Von Klein; Carl H. (1905). The Medical Features of the Papyrus Ebers. Chicago: Press of the American Medical Association. . 8 Nunn; John (2002). Ancient Egyptian Medicine. (p 151) University of Oklahoma Press. 9 Laurence, M. V. (2009). Hippocratic Recipes: Oral and Written Transmission of Pharmacological Knowledge in Fifth and Fourth Century Greece. (p 125) Leiden and Boston: Brill. 10 Hankinson, R. J. (2008). "Therapeutics- Van Der Eijk". The Cambridge Companion to Galen. Cambridge: Cambridge University Press. ISBN 978-1-139-82691-4. 11 "A History of Herbalism for Herbalists, Part 1: How the Arabs Saved Greek Sciences". Www.planetherbs.com. Last visited: 24 Jan. 2017. 12 Christopher Hobbs "History of Western Herbalism" Www.christopherhobbs.com. Last visited: 24 Jan. 2017. 13 Kyle, R. A.; Shampo, M. A. J. Am. Med. Assoc. 1979, 241(15), 1584. 14 Backer, H. J. Dutch Journal of Medicine 1943, 87, 1274–1278. 15 Courtwright; D. Forces of habit: drugs and the making of the modern world (1st ed, pp 36-37). Cambridge, Mass: Harvard University Press, (2001). 16 Luch A. (2009). Molecular, clinical and environmental toxicology. (1st ed., p 20). Springer. 17 Woodward, R. B.; Doering, W. E. J. Am. Chem. Soc. 1944, 66, 849-849. 18 "Quinine Sulfate". The American Society of Health-System Pharmacists. Last visited: 24 Jan. 2017. 19 Esu, E.; Effa, E. E.; Opie, O. N.; U waoma, A.; Meremikwu M. M. Cochrane Database of Systematic Reviews 2014, 9. Art. No.: CD010678. DOI: 10.1002/14651858.CD010678. 20 Buchner, A. Repertorium für die Pharmacie 1828, 29, 405–420. 21 Vane, J. R.; Bakhle, Y. S.; Botting, R. M. Annu. Rev. Pharmacol. Toxicol 1998, 38, 97–120. 22 Fleming, A. Br. J. Exp. Pathol. 1929 10(31), 226–236. 23 Henry, C. Chem. Eng. News 2005, 83(25), 97-97. 24 Tata, J.R. EMBO Reports 2005, 6(6), 490–6. 25 Wilson, C. O.; Gisvold O.; Doerge R. F. (1971) Wilson and Gisvold's Textbook of Organic Medicinal and Pharmaceutical Chemistry. (6th ed, p 1-4). J. B. Lippincott, Toronto, Canada. 26 Curran, D. P.; Liu, H. J. Am. Chem. Soc. 1992, 114, 5863- 5864. 27 Comins, D. L.; Baevsky, M. F.; Hong, H. J. Am. Chem. Soc. 1992, 114, 10971 -10972. 28 Takimoto, C. H.; Calvo, E. (2008). Principles of Oncologic Pharmacotherapy. (11th ed) Pazdur, R., Wagman, L. D.; Camphausen, K. A.; Hoskins, W. J. (Eds). 29 Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. I.; Sim, G.A. J. Am. Chem. Soc 1966, 88(16), 3888–3890. 30 WALL, M. E.; Wani M. C. Cancer Res. 1995, 55(4), 753–60. 31 Wani, M. C.; Taylor, H.; Wall, M. E.; Coggon, P.; McPhail, A. L. J. Am. Chem. Soc. 1971, 93(9),

Part 1: Introduction

2325–2327. 32 Holton, R. A.; Somoza, C.; Kim, H. B., Liang, F.; Biediger, R. J.; Boatman, P. D.; Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S., Zhang, P.; Murthi, K. K.; Gentile, L. N.; Liu, J. H. J. Am. Chem. Soc. 1994, 116(4), 1597–1598. 33 Holton, R. A.; Kim, H. B.; Somoza, C.; Liang, F.; Biediger, R. J.; Boatman, P. D.; Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K. K.; Gentile, L. N.; Liu, J. H. J. Am. Chem. Soc. 1994, 116(4), 1599–1600. 34 Stromgaard, K.; Krogsgaard-Larsen, P.; Madsen U. (2002). Textbook of Drug Design and Discovery. (1st Ed.). Taylor & Francis, Washington, DC. 35 Reynolds, C. H.; Merz, K. M.; Ringe, D. (2010). Drug Design: Structure- and Ligand-Based Approaches (1st Ed.). Cambridge, UK: Cambridge University Press. 36 Clement, O. O.; Mehl, A. T. (2000) HipHop: Pharmacophore based on multiple common-feature alignments, in Pharmacophore Perception, Development, and Use in Drug Design, (p 69-84) Guner OF ed, IUL, San Diego. 37 Reynolds, C. H.; Merz, K. M.; Ringe, D.; Tropsha, A. (2010). "QSAR in Drug Discovery". In Drug Design: Structure- and Ligand-Based Approaches (1st ed., p 151–164). Cambridge, UK: Cambridge University Press. 38 Leach, Andrew R.; Jhoti, H. (2007). Structure-based Drug Discovery. Berlin: Springer. 39 Mauser, H.; Guba, W. Curr. Opin. Drug Discovery J. 2008, 11(3), 365–374. 40 Greer, J.; Erickson, J. W.; Baldwin, J. J.; Varney, M. D. J. Med. Chem. 1994, 37, 1035–1054. 41 Evans, B. E.; Rittle, K. E.; Bock, M. G.; Di-Pardo, R. M.; Freidinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.; Anderson, P. S.; Chang, R. S.; Lotti, V. J.; Cerno, D. J.; Chen, T. B.; Kling, P. J.; Kunkel, K. A.; Springer, J. P.; Hirshfield, J. J. Med. Chem. 1988, 31(12), 2235–2246. 42 Wermuth, C. G.; J. Heterocycl. Chem. 1998, 35, 1091–1100. 43 Ripka, W. C.; De Lucca, G. V.; Bach, A. C. II; Pottorf, R. S.; Blaney, J. M. Tetrahedron 1993, 49, 3593-3608. 44 Hajduk, P. J.; Bures, M.; Praestgaard, J.; Fesik, S. W. J. Med. Chem. 2000, 43, 3443-3447. 45 Thompson, L. A.; Ellman, J. A.; Chem. Rev. 1966, 96, 555–600. 46 Horton, D. A.; Bourne, G. T.; Smythe, M. L. Chem. Rev. 2003, 103, 893-930. 47 Bunin, B.A. and Ellman, J. A. J. Am. Chem. Soc. 1992, 114, 10997–10998. 48 (a) Nicolaou, K. C.; Pfefferkorn, J. A.; Roecker, A. J.; Cao, G. Q.; Barluenga, S.; Mitchell, H. J. J. Am. Chem. Soc. 2000, 122, 9939-9953. (b) Nicolaou, K. C.; Pfefferkorn, J. A.; Mitchell, H. J.; Roecker, A. J.; Barluenga, S.; Cao, G. Q.; Affleck, R. L.; Lillig, J. E. J. Am. Chem. Soc. 2000 122, 9954-9967. (c) Nicolaou, K. C.; Pfefferkorn, J. A.; Barluenga, S.; Mitchell, H. J.; Roecker, A. J.; Cao, G. Q. J. Am. Chem. Soc. 2000, 122, 9968-9976. (d) Nicolaou, K. C.; Evans, R. M.; Roecker A. J.; Hughes, R.; Downes, M.; Pfefferkorn, J. A. Org. Biomol. Chem. 2003, 1, 908-920. 49 a) Norman, T. C.; Gray, N. S.; Koh, J. T.; Schultz, P. G. J. Am. Chem. Soc. 1996, 118, 7430-7431. b) Ding, S.; Gray, N. S.; Ding, Q.; Schultz, P. G., Tetrahedron Lett. 2001, 42, 8751-8755. c) Brun, F.; Legraverend, M.; Grierson, D. S. Tetrahedron Lett. 2001, 42, 8161-8164. 50 a) Abrous, L.; Hynes, J. Jr.; Friedrich, S. R.; Smith, A. B. III; Hirschmann, R. Org. Lett. 2001, 3, 1089-1092. b) Nilsson, J. W.; Thorstensson, F.; Kvarnström, I.; Oprea, T.; Samuelsson, B.; Nilsson, I.; J. Comb. Chem. 2001, 3, 546-553. c) Lewell, X. Q.; Judd, D. B.; Watson, Ś. P.; Hann, M. M. J. Chem. Inf. Comput. Sci. 1998, 38, 511-522. d) Mason, J. S.; Morize, I.; Menard, P. R.; Cheney, D. L.; Hulme, C.; Labaudiniere, R. F. J. Med. Chem. 1999, 42, 3251-3264.

Part 1: Introduction

51 Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev. 2001, 46(1-3), 3- 26. 52 Sternbach, L. H. J. Med. Chem. 1979, 22, 1-7. 53 Herpin, T. F.; Van Kirk, K. G.; Salvino, J. M.; Tang Yu, S.; Labaudinière, R. F. J. Comb. Chem. 2000, 2, 513-521. 54 Grasso, S.; De Sarro, G.; De Sarro, A.; Micale, N.; Zappalà, M.; Puia, G.; Baraldi, M.; De Micheli, C. J. Med. Chem. 1999, 42, 4414-4421. 55 a) Richter, P. H.; Morgenstern, O. Pharmazie 1984, 39, 301-314. b) Morgenstern, O.; Richter P. H. Pharmazie 1992, 47, 655-677; c) Morgenstern, O. Pharmazie 2000, 55, 871-891. 56 a) Osada, H.; Ishinabe, K.; Yano, T.; Kajikawa, K.; Isono, K. Agric. Biol. Chem. 1990, 54, 2875- 2881.; b) Osada, H.; Uramoto, M.; Uzawa, J.; Kajikawa, K.; Isono, K. Agric. Biol. Chem. 1990, 54, 2883- 2887.

Part 1: Introduction

II. COMBINATORIAL CHEMISTRY

1. DEFINITION&PRINCIPLES

The introduction of a new pharmaceutical is a lengthy and expensive undertaking. Methods which promise to shorten the time or the cost are eagerly applied, as has been the case with combinatorial chemistry. Combinatorial chemistry is a collection of methods that allow the simultaneous chemical synthesis of large numbers of compounds using a variety of starting materials, instead of doing reactions in a stepwise, time-consuming manner (figure II.1). The resulting combinatorial library can contain all of the possible chemical structures that can be produced by combining different building blocks (figure II.2. e.g. n x m compounds of type A-B from n building blocks A and m building blocks B)

A1 + B1 A1B1

A1 + B2 A1B2 A + B A B 1 3 1 3

Figure II.1 Conventional synthesis.

A1 B1 A1B1 A2B1 An-1B1 AnB1

A2 B2 A1B2 A2B2 An-1B2 AnB2 +

An-1 Bm-1 A1B3 A2Bm-1 An-1Bm-1 AnBm-1

An Bm A1B1 A2Bm An-1Bm AnBm

Figure II.2 Combinatorial Synthesis.

Libraries can be constructed in solution or on solid supports and the choice between these techniques is often a matter of personal preference, and the task at hand.

2. HISTORY OF COMBINATORIAL CHEMISTRY

Combinatorial chemistry grew out of peptide chemistry and the path leading to the present state of combinatorial chemistry essentially started with the solid phase synthetic experiments on peptides by Bruce Merrifield in 19631,2. After coupling the first amino acid covalently onto an insoluble

Part 1: Introduction polymer resin, the peptide was elongated while remaining attached to the solid support: In contrast to classical solution synthesis of peptides, problems due to solubility/aggregation issues were completely eliminated.

Because of the essentially iterative reactions and its susceptibility to automation, a dramatic increase of product output was achievable in a given amount of time. Moreover, by the use of reagents in excess and the ease of removing these reagents and byproducts from the resin-bound intermediates by simple washing ensured quick and high yielding access to final peptide products. At first this extremely useful technology was employed in a parallel fashion.

It was Furka in 1988 who first realized that the methodology could lead to simultaneous synthesis of large collections of peptides and conceived of the split-mix method or (“Split-pool” method) 3, the procedure being depicted in figure II.3. The synthesis is executed by repetition of the following three simple operations that form a cycle: 1. Dividing the solid support into equal portions; 2. Coupling each portion individually with only one of the different amino acids; 3. Mixing and homogenizing the portions. By repeating this cycle several times using sets of selected amino acids, large libraries can be obtained; however each bead has just one unique product attached to it, leading to a so called “ one bead-one compound” library.

After carrying out the synthesis of the resin-bound peptide library, all peptides can be cleaved from the support to yield the mixture of free peptides in solution, also called soluble peptide libraries. In such collections millions of peptides may be present and identifying a bioactive peptide among them seems like finding a needle in a hay stack. Nevertheless, appropriate strategies have been developed to address the problem, such as deconvolution strategies or tag-based library synthesis; however they can be lengthy and time consuming.

Part 1: Introduction

A1 A2 Ai

A2 A A1 i

A2 A2 A2

Ai Ai A A1 A1 A1 i

B1 B2 Bi

A2B1 A2B2 A2Bi

AiB1 A B A1B1 A1B2 i 2 A1B1 AiBi

A1B1 A1B2 A1Bi

A2B1 A2B2 A2Bi

A B A B A B i 1 i 2 i i

Figure II.3 “Split-pool” method.

Another common strategy to build up a combinatorial library is parallel synthesis. This term was introduced by Lam 4. In this method, the reactions are performed in spatially separated reaction vessels throughout the whole synthesis (figure II.4). In comparison with "split-pool" methods, parallel synthesis is less reaction efficient and therefore the generated libraries are usually smaller,

Part 1: Introduction but each vessel now contains its own unique compound ("one vessel - one compound" principle). Another advantage is that parallel synthesis can be performed both in solution and on solid phase.

A1-4

A1 A1 A1 A1 A3 A3 A3 A3

A2 A2 A2 A2 A4 A4 A4 A4

B1-2

A1B1 A1B1 A1B2 A1B2 A3B1 A3B1 A3B2 A3B2

A2B1 A2B1 A2B2 A2B2 A4B1 A4B1 A4B2 A4B2

C1-2

A1B1C1 A1B1C2 A1B2C1 A1B2C2 A3B1C1 A3B1C2 A3B2C1 A3B2C2

A2B1C1 A2B1C2 A2B2C1 A2B2C2 A4B1C1 A4B1C2 A4B2C1 A4B2C2

Figure II.4 Parallel synthesis

Geysen introduced the first automated solid phase procedures and tools for peptide synthesis (Multipin apparatus), making the whole process technically simpler in 1984 and produced large scale compound collections of peptides using a pin shaped solid support5 (figure II.5). Houghton introduced “tea bag” methodologies in 1985 in which porous bags of resins were suspended in solutions of reagents6 (figure II.6).

Part 1: Introduction

a Pin

b Solution

Figure II.5 The multipin apparatus5

Tea-bag reaction vessels with bags

Figure II.6 The tea bag method6

3. SOLID-PHASE SYNTHESIS AS A TOOL FOR COMBINATORIAL CHEMISTRY

Solid phase synthesis is that branch of organic chemistry in which compounds are synthesized while they are covalently anchored to a polymeric carrier. It is highly advantageous for multistep iterative processes and notable for the comparative ease of purification by simple filtration which allows to drive reactions to completion by the use of excess reagents. Throughout the previous decade, solid phase organic synthesis (SPOS) has dominated combinatorial chemistry, and many novel methods have been developed as a result. Initially developed by Merrifield in the sixties for the synthesis of peptides7, it was later applied to other oligomers such as oligonucleotides8 and peptides9 and more recently introduced in the synthesis of non-oligomeric small molecules10, 11, 12. An advantage for the use in the combinatorial chemistry is the possibility to automate these solid phase syntheses. Automated systems with computer-controlled mechanical robots can routinely add the desired reagents and solvents accurately, subsequently let the reaction run at the desired temperature after which the resins are automatically filtered and washed13.

Part 1: Introduction

To perform an efficient solid-phase synthesis two elements have to be chosen rationally, the solid support matrix and the linker which connects the substrate and the solid-support via a connection point (figure II.7). A spacer, which creates a defined distance between the solid support and the linker to avoid sterical hindrance, can also be incorporated, but will not be discussed further as this element can be comprised in the linker.

Linker Moiety Substrate

Support Linker Attachment

Figure II.7 Schematic representation of a solid-phase structure.

3.1. Solid Support

Since Merrifield introduced solid-phase peptide synthesis [SPPS], by using a chloromethylated polystyrene resin (lightly cross-linked with divinylbenzene, so called Merrifield resin II.1, figure II.8), the use of polymers to facilitate synthesis and product purification has become widespread and the resin itself underwent an evolution14. There are different types of solid supports, based on alternative polymeric networks. By far the most common polymer matrix is based on polystyrene which is crosslinked with p-divinylbenzene 1%. The individual resin beads have a size of around 75-150 μm and have a good mechanical stability, high loading capacity and are chemically inert under various reaction conditions while being relatively inexpensive.

They display good swelling properties in organic solvents such as THF, dichloromethane and DMF, but swell badly in water, methanol and hexane15. Another particular advantage is the ease of modifying the aromatic backbone with a variety of functional groups, which allows the chemist to attach diverse linkers onto the resin. These functional groups are not only present on the surface of the polymer particle, but are evenly distributed throughout the whole bead16. When the resin is nicely swollen in an appropriate solvent, the polymer backbone induces no steric hindrance at the reactive sites of the resin as illustrated by Sarin et al., who synthesized peptides up to 60 residues on solid phase without loss of efficiency compared to a similar solution phase protocol17.

Part 1: Introduction

Better swelling in protic polar solvents is possible with the use of so-called TentaGel resin18. The better compatibility of the resin with such solvents is a result of the grafting of more polar polyethylene glycol arms on a central polystyrene matrix. However, disadvantages of this type of resin, are the inevitable lower loading capacity and the more expensive purchase.

O O OH Cl

Cl HO O O

II.1 "Merrifield" resin II.2 "TentaGel" resin n=4

O O O O

NMe2 NH HN N NH2 H

NMe2 HN NMe2 O O O II.3 "Sheppard" resin

Figure II.8 Polymer backbones of classic solid phase resins

So-called Sheppard resin19 is built up from a polyacrylamide matrix and was developed specifically for peptide synthesis, as the high amide content allows better interaction with the growing peptide and thus preventing self-aggregation and thus lower reactivity of the resin-bound peptide. In addition to these more popular polymeric matrices, many (and are) alternative polymeric supports with interesting properties have been developed, but they are less routinely used by other groups, potentially because of their expensive purchase price or because they are not (yet) commercially available.

In this PhD work the popular polystyrene matrix-based solid carriers was chosen.

Part 1: Introduction

3.2. The Linker

The linker is often described as a bifunctional protecting group and requires careful consideration in view of the intended synthetic transformations to be applied on the solid phase. On one hand it is bound to the polymer matrix via a permanent covalent bond while on the other hand a temporary covalent anchoring element is used to attach the first building block to the solid support. The linker should fulfill a number of criteria: (i) stable against a planned set of conditions; (ii) immobilization of organic molecule should be readily achieved in high yield and (iii) synthesized molecules on polymer supports should be cleaved under mild conditions that do not degrade the products.

Linkers are categorized according to the method of cleavage from the resin20, and can be further sub-classified based on the liberated functional group of the product after cleavage (i.e. carboxylic acid, carboxamide, alcohol, amine and ketone linkers). The most commonly applied conditions for cleavage are acidic conditions and these are usually based on the formation of a resin-bound benzyl cation during cleavage. According to the relative stability of the latter, the cleavage conditions can range from strongly (HF, TFMSA) to mildly acidic (0.1% TFA).

The original Merrifield resin II.1, bearing no stabilizing substituents, needed to be treated with HF to cleave off the desired peptide. 4-Alkyloxybenzyl linker (Wang resin II.4) allows the use of a concentrated TFA solution to cleave off the desired product21. With additional methoxysubstituents on one22 (SASRIN resin II.5) or both23 (HAL resin II.6) of the ortho- positions of the benzylic moiety, even dilute TFA solutions can be used to induce cleavage. Another strategy to stabilize the cation consisted of the introduction of one or two (substituted) phenyl groups on the benzylic positions, resulting respectively in benzhydryl24 (Rink resin II.7) or trityl25 type resins (trityl resin II.8 or 2-chlorotrityl II.9). Both linkers allow cleavage of the desired products in dilute TFA solutions. More details about linkers, their applications and cleavage conditions will not be discussed further in this work due to the broad scope of this topic, but are extensively reviewed in the literature26. Many alternative linker systems are designed and for a comprehensive overview it is better to refer to the literature20.

Part 1: Introduction

R1 II.4; Wang, R1=H, R2=H Cl O II.5; SASRIN, R1=OMe, R2=H II.6; HAL, R =OMe, R =OMe OH 1 2 R II.1: Merrifield 2

O Cl II.8; trityl, R=H R II.9; 2-chlorotrityl, R=Cl

II.7; Rink OMe H2N MeO

Figure II.9 Resins with a varying stability towards acids

4. ANALYSIS OF SOLID PHASE INTERMEDIATES

Monitoring techniques used in solution chemistry (e.g. TLC) are not possible to apply on solid phase syntheses due to the physical nature of the resin beads. Although the resin-bound intermediates can in principle be cleaved from the resin and thus submitted to the conventional analytical techniques, it is important to understand that this does not allow "real time reaction monitoring" as the resin-cleavage already takes some time. Moreover, it has to be kept in mind that this is a destructive analytical approach, as cleaved intermediates can no longer be recovered for further use in the solid phase synthesis sequence. Therefore, new techniques were developed or adapted from existing techniques.

4.1. On-bead analysis methods

On-bead analysis using classical NMR27 techniques has limitations, due to insoluble character of the polymer matrix which leads to broad proton signals in 1H NMR-spectroscopy. This so-called "Gel-phase NMR" technique is quasi only feasible on resin matrices containing more flexible chains (TentaGel instead of polystyrene resins)28. A more advanced methodology is HR-MAS NMR (High Resolution-Magic Angle Spinning NMR), where the sample is rotated at a predetermined angle (54.7°, the "magic" angle) with respect to the applied magnetic field, giving better resolution 29. However, such MAS probes are expensive, which makes this technique less in general use.

Part 1: Introduction

On-resin reaction monitoring via FT-IR30 techniques is also possible, but requires the appearance or disappearance of a characteristic absorption band in the spectrum related to the transformation of a functional group. In addition, band overlap with functional groups present on the solid support could also hinder an accurate monitoring of the reaction.

The use of color tests31 allows a fast and cheap alternative consuming only tiny amounts of resin- bound product. The use of colorimetric functional group tests, wherein aliquots of resin are mixed with stock solution of coloring agents and changes in solution/resin color are used to indicate the presence or absence of functional groups on the resin. Most color tests have been developed for the determination of free amine groups32, but there are also color tests designed for other functional groups such as alcohols33, thiols34, aldehydes35 and carboxylic acids36. A short overview of the tests applied during this research is depicted in figure II.10.

O primary or secondary amine O R Cl Cl Cl N NHR Chloranil test Cl Cl acetaldehyde Cl Cl O [via initial enamine formation] II.10 O II.11

SO3H HN primary amine TNBS test O2N NO2 O2N NO2

NH2

NO2 NO2 II.12 II.13

Figure II.10 Color tests for solid-supported amines as applied during this research

4.2. UV quantification of resin cleavable chromophore groups – the Fmoc test

The use of cleavable resin bonded chromophore groups makes more quantitative data available. UV spectrophotometric determination of the concentration of the respective cleaved chromophore can be correlated to the absolute amount of resin-bound product.

A more frequently used method for quantitative measurements of coupling efficiencies is Fmoc UV-quantification. This method, developed by Sheppard37, is therefore only applicable when using an Fmoc-based synthetic strategy. It is based upon the formation of the chromophore dibenzofulvene-piperidine adduct II.18 after base-induced elimination of the Fmoc-protecting

Part 1: Introduction group with piperidine (figure II.11). However, because of inconveniences concerning the ordering of piperidine and the toxicity problems related, we have used 4-methylpiperidine as suggested by Hachmann as a suitable replacement38.

O H N O "E1cb" O H R O N H II.15 CO2 N H N II.14 Me II.16 Me  =300nm Me max,1 Figure II.11 Fmoc-deprotection mechanism and 4-methylpiperidine-dibenzofulvene adduct formation.

39 By measuring the absorption at 300 nm (휆max,1, figure II.12), the concentration of this adduct can be determined using the Lambert-Beer equation: A = ε.l.c (in which A = absorption, ε = molar extinction coefficient, l = path length, c = molar concentration).

Part 1: Introduction

Figure II.12 UV spectrum of a solution containing the dibenzofulvene-(4-methylpiperidine) adduct

Practically, the UV absorbance of the adduct is measured at 300 nm, while the extinction coefficient of the 4-methylpiperidine-dibenzofulvene adduct was determined using a calibration line by treating different concentration of Fmoc-alanine.

For the two spectrophotometers used during the work, values of 8.3585 M-1cm40 and 8.4137 M- 1cm-1 were found respectively, for concentrations between 0 and 0.16 mM.

There have been several other useful chromophores reported in solid phase synthesis. Amines protected with the Bsmoc-41 or o-NBS group42 can be used in an analogous manner. Using DMT- Cl43 or NPIT44, leads to a resin-bound (dimethoxy)trityl group, which can be quantified as dimethoxytritylkation after acid treatment. As an example of another functional group, aldehydes can be determined in an analogous manner after derivatization using dansylhydrazine45 or the fluorenylmethyl ester of 4-hydrazinobenzoic acid46.

Part 1: Introduction

References

1 Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149. 2 Merrifield, R. B. Science 1986, 232, 341. 3 a) Furka, A. A.; Sebesteyn, F.; Asgedom, M.; Dibo, G. Int. J. Pept. Protein Res. 1991, 37(6), 487-493. b) Sebesteyn, F.; Dibo, G.; Kovacs, A.; Furka, A. Bioorg. Med. Chem. Lett. 1993, 3(3), 413-418. c) Furka, A. A. Drug Discov. Today 2002, 7, 1. 4 Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82-84. 5 a) Geysen, H. M.; Meloen, R. H.; Barteling, S. J. Proc. Natl. Acad. Sci. 1984, 81(13), 3998-4002. b) Geysen, H. M.; Barteling, S. J.; Meloen, R. H. Proc. Natl. Acad. Sci. 1985, 82(1), 178 182. 6 Houghten, R. A. Proc. Natl. Acad. Sci. 1985, 82(15), 5131-5135. 7 Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. 8 a) Letsinger, R.L .; Mahadevan, V. J. Am. Chem. Soc. 1965, 87, 3526-3527. b) Elmblad, A.; Josephson, S.; Palm, G. Nucleic Acid Res. 1982, 10, 3291-3301. 9 a) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am. Chem. Soc. 1992, 114, 10646- 10647. b) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D. A.; Banvill, S. C.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K.; Spell Meyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen, F. E.; Bartlett, P. A. Proc. Natl. Acad. Sci. USA, 1992, 89, 9367- 9371. c) Krujtzer, J. A.; Hofmeyer, L. J.F.; Heermabrug, W.; Versluis, C.; Liskamp, R. M. J. Chem. Eur. J. 1998, 4, 1570-1580. 10 Frechet, J. M. J. Tetrahedron 1981, 37, 663-683. 11 Bunin, B. A.; Ellman, J. A. J. Am. Chem. Soc. 1992, 114, 10997-10998. 12 Thompson, L. A.; Ellman, J. A. Chem. Rev. 1996, 96, 555-600. 13 Weber, A.; von Roedern, E.; Stilz, H. A. J. Comb. Chem. 2005, 7, 178-184. 14 Merrifield, R. B. Solid Phase Peptide Synthesis. In Gutte, B. "Peptides: Synthesis, Structures and Applications" 1995. (pp. 94-170). London: Academic Press Limited. 15 Santini, R.; Griffith, M. C.; Qi, M. Tetrahedron Lett. 1998, 39(49), 8951-8954. 16 a) Kress, J.; Rose, A.; Frey, J. G.; Brocklesby, W. S.; Ladlow, M.; Mellor, G. W.; Bradley, M. Chem. Eur. J. 2001, 7(18), 3880-3883. b) Rademann, J.; Barth, M.; Brock, R.; Egelhaaf, H. J.; Jung, G. Chem. Eur. J. 2001, 7(18), 3884- 3889. 17 Sarin, V. K.; Kent, S. B. H.; Mitchell, A. R.; Merrifield, R. B. J. Am. Chem. Soc. 1984, 106(25), 7845- 7850. 18 Wright, P.; Lloyd, D.; Rapp, W.; Andrus, A. Tetrahedron Lett. 1993, 34, 3373-3376. 19 a) Atherton, E.; Clive, D. L. J .; Sheppard, R. C. J. Am. Chem. Soc. 1975, 97, 6584-6585; b) Arshady, R.; Atherton, E.; Clive, D. L. J.; Sheppard, R.C. J. Chem. Soc. Perkin Trans. 1981, 1, 529-537; c) Atherton, E.; Logan, C. J.; Sheppard, R. C. J. Chem. Soc. Perkin Trans. 1981, 1, 538-546; d) Kanda, P.; Kennedy, C.; Sparrow, J. T. Int. J. Pept. Protein Res. 1991, 38, 385-391. 20. a) James, I. W. Tetrahedron 1999, 55, 4855-4946. b) Guillier, F.; Orain, D.; Bradley, M. Chem. Rev. 2000, 100, 2091-2157. c) Comely, A. C.; Gibson, S. E. Angew. Chem. Int. Ed. 2001, 40, 1012-1032. 21 Wang, S. S. J. Am. Chem. Soc. 1973, 95(4), 1328-1333. 22 Mergler, M.; Tanner, R.; Gosteli, J.; Grogg, P. Tetrahedron Lett. 1988, 29(32), 4005-4008. 23 Albericio, F.; Barany, G. Tetrahedron Lett. 1991, 32(8), 1015-1018. 24 Rink, H. Tetrahedron Lett. 1987, 28(33), 3787-3790. 25 Barlos, K.; Gatos, D.; Kallitsis, J.; Papaphotiu, G.; Sotiriu, P.; Wenging, Y.; Schäfer, W. Tetrahedron

Part 1: Introduction

Lett. 1989, 30(30), 3943-3946. 26 a) James, I. W. Tetrahedron 1999, 55(16), 4855-4946. b) Jung, G., Combinatorial Chemistry: Synthesis, Analysis, Screening 1999. (pp. 167-228). Weinheim, Germany: Wiley-VCH. 27 a) Gordeev, M. F.; Patel, D. V.; Gordon, E. M. J. Org. Chem. 1996, 61(3), 924-928. b) Garigipati, R. S.; Adams, J. L.; Sarkar, S. K. J. Org. Chem. 1996, 61(8), 2911-2914. c) Riedl, R.; Tappe, R.; Berkessel, A. J. Am. Chem. Soc. 1998, 120(35), 8994-9000. 28 a) Giralt, E.; Rizo, J.; Pedroso, E. Tetrahedron 1984, 40, 4141-4152 ; b) Look, G. C.; Holmes, C. P.; Chinn, J. P.; Gallop, M. A. J. Org. Chem. 1994, 59, 7588-7590 ; c) Lorgé, F.; Wagner, A.; Mioskowski, C. J. Comb. Chem. 1999, 1, 25-27. 29 a) Fitch, W. L.; Detre, G.; Holmes, C. P.; Shoolery, J. N.; Keifer, P. A. J. Org. Chem. 1994, 59, 7955- 7956; b) Anderson, R. C.; Jarema, M. A.; Shapiro, M. J.; Stokes, J. P.; Ziliox, M. J. Org. Chem. 1995, 60, 2650-2651; c) Warras, R.; Wieruszeski, J. M.; Lippens, G. J. Am. Chem. Soc. 1999, 121, 3787-3788 ; d) Pursch, M.; Schlotterbeck, G.; Tseng, L. H.; Albert, K.; Rapp, W. Angew. Chem. Int. Ed. Engl. 1996, 35, 2867-2869. 30 a) Yan, B.; Fell, J. B.; Kumaravel, G. J. Org. Chem. 1996, 61(21), 7467-7472. b) Yan, B.; Kumaravel, G. Tetrahedron 1996, 52(3), 843-848. c) Chan, T. Y.; Chen, R.; Sofia, M. J. Tetrahedron Lett. 1997, 38(16), 2821-2824. 31 a) Vázquez, J.; Qushair, G.; Albericio, F. Methods Enzymol. 2003, 369, 21-35; b) Gaggini, F.; Porcheddu, A.; Reginato, G; Rodriguez, M.; Taddei, M. J. Comb. Chem. 2004, 6, 805-810. 32 a) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem. 1970, 34, 595-598. b) Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 1981, 117, 147-157. c) Hancock, W. S.; Battersby, J. E. Anal. Biochem. 1976, 71, 260-264. d) Krchnak, V.; Vagner, J.; Safar, P.; Lebl, M. Collect. Czech. Chem. Commun. 1988, 53, 2542-2548. e) Reddy, M. P.; Voelker, P. J. Int. J. Peptide Protein Res. 1988, 31, 345-348. f) Vojkovsky, T. Pept. Res. 1995, 8(4), 236-237. g) Madder, A.; Farcy, N.; Hosten, N. G. C.; De Muynck, H.; De Clercq, P. J.; Barry, J.; Davis, A. P. Eur. J. Org. Chem. 1999, 2787-2791. h) Mařı,́ k. J.; Song, A.; Lam, K. S. Tetrahedron Lett. 2003, 44(23), 4319-4320. i) Blackburn, C. Tetrahedron Lett. 2005, 46(9), 1405-1409. j) Claerhout, S.; Ermolat'ev, D. S.; Van der Eycken, E. V. J. Comb. Chem. 2008, 10(4), 580-585. 33 a) Kuisle, O.; Lolo, M.; Quiñoá, E.; Riguera, R. Tetrahedron 1999, 55(51), 14807-14812. b) Attardi, M. E.; Falchi, A.; Taddei, M. Tetrahedron Lett. 2000, 41(38), 7395-7399. c) Attardi, M. E.; Taddei, M. Tetrahedron Lett. 2001, 42(15), 2927. d) Burkett, B. A.; Brown, R. C. D.; Meloni, M. M. Tetrahedron Lett. 2001, 42(33), 5773-5775. e) Caroen, J.; Van der Eycken, J. Tetrahedron Lett. 2009, 50(1), 41-44. 34 a) Virgilio, A. A.; Ellman, J. A. J. Am. Chem. Soc. 1994, 116(25), 11580-11581. b) Badyal, J. P.; Cameron, A. M.; Cameron, N. R.; Coe, D. M.; Cox, R.; Davis, B. G.; Oates, L. J.; Oye, G.; Steel, P. G. Tetrahedron Lett. 2001, 42(48), 8531-8533. 35 a) Cournoyer, J. J.; Kshirsagar, T.; Fantauzzi, P. P.; Figliozzi, G. M.; Makdessian, T.; Yan, B. J. Comb. Chem 2002, 4(2), 120-124. b) Shannon, S. K.; Barany, G. J. Comb. Chem. 2004, 6(2), 165-170 c) Vázquez, J.; Albericio, F. Tetrahedron Lett. 2001, 42(38), 6691-6693. 36 Attardi, M. E.; Porcu, G.; Taddei, M. Tetrahedron Lett. 2000, 41(38), 7391-7394. 37 Atherton, E.; Sheppard, R. C.; In Solid Phase Peptide Synthesis: A Practical Approach, 1989, (pp. 1- 203). Oxford, England: IRL Press. 38 Hachmann, J.; Lebl, M. J. Comb. Chem. 2006, 8, 149.

39 The absorbance can also be measured at λmax,2 = 290 nm.

Part 1: Introduction

40 Caroen J., Ontwikkeling van een vastefasesynthesestrategie voor 1,2,3,4,5,6-hexahydro-1,5- benzodiazocine-2,6-dionen voor toepassing in combinatorische bibliotheken, (Development of a solid- phase synthesis strategy for 1,2,3,4,5,6-hexahydro-1,5-benzodiazocine-2,6-diones for application in combinatorial libraries) 2012. PhD dissertation, UGent. 41 Bsmoc = 1,1-dioxobenzo[b ]thiophene-2-ylmethyloxycarbonyl : Carpino, L. A.; Ismail, M.; Truran, G. A.; Mansour, E. M. E.; Iguchi, S.; Ionescu, D.; El-Faham, A.; Riemer, C.; Warrass, R. J. Org. Chem. 1999 , 64, 4324-4338. 42 o-Ns (of o-NBS) =2-nitrobenzeensulfonyl: Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1998, 120, 2690-2691. 43 DMT-Cl =4,4’-dimethoxytritylchloride: Reddy, M. P.; Voelker, P. J., Int. J. Peptide Protein Res. 1988, 31, 345-348. 44 NPIT =nitrophenylisothiocyanate-O-trityl: Chu, S. S.; Reich, S. H. Bioorg. Med. Chem. Lett. 1995, 5, 1053-1058. 45 Yan, B.; Liu, L.; J. Org. Chem. 1997, 62, 9354-9357. 46 Shannon, S. K.; Barany, G.; J. Org. Chem. 2004, 69, 4586- 4594.

Part 1: Introduction

III LITERATURE REVIEW ON BENZODIAZOCINE DERIVATIVES

III.1 INTRODUCTION Benzene-annelated medium ring diaza-compounds are an important class of heterocyclic structures, occurring in a wide range of biologically active products1-3. There is a sound basis for their appeal in pharmacological research; they offer restricted conformational flexibility, allowing higher intrinsic binding capacity because of favorable entropic reasons while their moderate size allows the introduction of diverse side chains to obtain compounds complying to Lipinski’s rule of five4. As such they possess properties of so-called privileged structures, as evidenced by the pharmacologically successful 1,4-benzodiazepine archetype (see chapter I) 5, 6. The term ‘medium ring’ by origin is applied to alicyclic compounds having a ring size in the range of 7-117, although more commonly designated to 7-9 membered cycles. By far the most reported medium-ring derivatives are seven-membered systems, as evidenced by naturally occurring carbacycles (for example Frondosin B III.18 isolated from the sponge Dysidea frondosa, and Laserpitine III.29 which has been isolated from underground parts of Laserpitium latifolium (Apiaceae) species and synthetical azepines and derivatives such as benzodiazepines (for example Diazepam III.310 which is used in treatment of anxiety).

CH3 H3C O CH3 O HO H3C CH3 O O H3C N CH 3 H OH H C 3 O CH3 Cl N HO CH3 O

O CH3 H3C Frondosin B Laserpitin Diazepam (Valium) III.1 III.2 III.3 Figure III.1 Examples of naturally occurring 7-membered carbacycles and synthesized benzodiazepines.

Part 1: Introduction

Much less data is available on the homologous eight-membered ring structures, although examples are known with interesting biological activity. For example, benzodiazocines have been shown to display diverse activities by acting as central nervous system depressant (III.4)11, muscle relaxant (III.5) and (III.6)12,13, RNA polymerase inhibitor (III.7)14, Ca2+-sensitizer (III.8) 15, inhibitor of 16 17 leukemia cells (III.9) and activator for PKCε (III.10) . Additionally, some members of the benzodiazocine system have found application as homologues of benzodiazepine drugs (III.11)18 (figure III.1) and as reversal agents in multidrug resistance.19,20.

N NH O O HN HN N NH HO

R N

III.4 III.5 R = H III.7 III.6 R = CH3

O O H O O NH H N N H3C N N Me N OH Cl N O N

C10H21-n

Benzolactam-V8-310 (+)-CGP 48506 (BL-V8-310)

III.8 III.9 III.10 III.11

Figure III.1. Examples of biologically active benzodiazocines.

The dibenzodiazocine skeleton has gained considerable interest as a challenging target and pharmacologically interesting compounds have been identified, such as the 17-β-hydroxysteroid dehydrogenase type 3 inhibitor (III.12)21 and the cholesterol-lowering III.1322.

Part 1: Introduction

In recent years, material chemists have explored the electrochemical properties of diaryldibenzo[b,f][1,5]diazocines (III.14) which were found to be useful as a basis for molecular machines and artificial muscles23,24. Moreover, a bridged variant of dibenzo[b,f][1,5]diazocine, “Troger’s base” (III.15) has a chiral aromatic cleft structure with a unique ability to host guests and as a result, derivatives can act as a molecular receptor (figure III.2)25,26.

O Cl N N N Me N

Me HN N N N Cl Troger,s base

III.12 III.13 III.14 III.15

Figure III.2 Examples of interesting dibenzodiazocines.

However, medium sized heterocycles, (especially eight- or higher membered rings) are difficult to prepare due to enthalpic and entropic reasons and potential trans-annular interactions27. In continuation of our interest in developing expeditious routes to various medium-sized heterocyclic molecules, our laboratory is interested in establishing a convenient synthetic route to diverse 1,5- benzodiazocine-2,6-diones III.16 (figure III.3) for potential pharmaceutical applications.

R10R1 O N R2 R9 R3 R R8 N 4 R5 R7 O R6

III.16 Figure III.3 Target molecules: 1,5-benzodiazocine-2,6-diones III.16. As any recent overview on benzodiazocines is lacking in the literature28, in this chapter representing syntheses of the different possible skeletons of benzodiazocines (1,2-; 1,3-; 1,4-; 1,5-

Part 1: Introduction

; 1,6-; 2,3-; 2,4-; 2,5-; 3,4-) and available data regarding their biological activity are reviewed, with particular comprehensive focus on reported cases of our target skeleton (1,5-benzodiazocine-2,6- dione III.16).

III.2 1,2-BENZODIAZOCINES

1,2-Benzodiazocines are a class of azobenzenes which are the most frequently used photochromic switches in chemistry29 and they are mainly presented as dibenzo[1,2]-diazocines. Merino et. al30 reported the synthesis of 3,3`-amino-substituted III.19 and 3,3`-acetamide-substituted diazocine derivatives III.20; starting from commercially available 1,2-bis(4-aminophenyl)ethane III.17, a nitration reaction gives 1,2-bis(2-nitro-4-aminophenyl)ethane III.18, for which intramolecular reductive coupling of the nitro groups give azo compound III.19. The acetamide derivative III.20 is formed by treatment of 3,3`-diamino-2,2`-ethylene-bridged azobenzene (3,3`-diamino-EBAB) III.19 with acetic anhydride (figure III.4).

NO2 NO2 NaNO3, H2SO4 40°C, 99% H2N NH2 H2N NH2

III.17 III.18

glucose, NaOH EtOH, H2O, 30%

Ac2O, 100% HN N N NH H2N N N NH2 O O

III.20 III.19

Figure III.4 Synthesis of 3,3`-diamino-EBAB III.19 and its acetamide derivative III.20.

Part 1: Introduction

III.3 1,3-BENZODIAZOCINES

The first compounds of this series have been synthesized31,32 by Kempter through reduction of 3- (2-nitrophenyl)propionaldehyde oxime derivatives (III.21) to afford (III.22) which undergo cyclocondensation with formaldehyde, phosgene or carbon disulfide to give a variety of 1,3- benzodiazocines III.23 (figure III.5). A preference for the α-adreno-receptors was observed with 33 moderate seven fold α2/α1 selectivity .

X NO 2 H NH2 HN 2 CH2O or COCl2 or CS2 R1 R1 NH N NH OH 2 R2 R2 R2 R1 III.21 III.22 III.23

R1 = H, Me X = H2, O, S R2 = H, Me, Ph R1 = H, Me R2 = H, Me, Ph

Figure III.5 The first synthesized 1,3-benzodiazocines in 1978 by Kempter.

The 1,3-benzodiazocine scaffold was patented in 198634. The cyclization step involves condensation of the α,ω-ureido-aniline III.24 with carbon disulfide in hot ethanol affording the thioureas III.25 (figure III.6).

O R N N H H H N CS2/ EtOH N R NH2 HN Reflux S O 67%; R= cyclohexyl 72%; R= phenyl III.25 III.24 63%; R= 2-chlorophenyl 74%; R= 2-aminophenyl

Figure III.6 Synthesis of thioxobenzo[d][1,3]diazocine-3(4H)-carboxamide derivatives.

Part 1: Introduction

III.4 1,4-BENZODIAZOCINES

III.4.1 1,4-Benzodiazocine-2,5-diones via ring expansion (Muchowski)

The first publication on the 1,4-benzodiazocine skeleton describes a three step synthesis of unsubstituted 1,4-benzodiazocine-2,5-dione III.3035. Acetylation of sodioisatin III.26 with azidoacetyl chloride produced azido compound III.27, which was catalytically hydrogenated in one step to the aminoacetyloxindole III.28. Extraction of an aqueous bicarbonate solution of III.28 with DCM gave the parent 1,4-benzodiazocine III.30 via ring expansion (figure III.7).

O O

O N3CH2COCl O H /Pd-C, 60 C O N- N 2 N Na+ HOAc, HClO CH2N3 4 CH NH HClO O O 2 2. 4

III.26 III.27 III.28

KHCO3

O OH NH NH N N H O O

III.30 III.29

Figure III.7 Synthesis of the parent 1,4-diazocine-2,5-dione (III.30) by Muchowski.

III.4.2 1,4-Benzodiazocine-2-ones via lactamization

Another synthetic route developed by Muchowski involved the synthesis of 1,4-benzodiazocines III.35 via the corresponding 1,4-benzodiazocin-2-ones III.34. Alkylation of secondary amine III.31 with ethyl bromoacetate III.32 was followed by 3 steps to obtain α,ω-aminocarboxylic acid III.33. Cyclization using DCC in pyridine or acetonitrile gave the lactams III.3436, which could be further reduced to the diamine III.35 (Figure III.8).

Part 1: Introduction

R NHR O N COOH + Br O NO2 NH2 III.31 III.32 III.33 a: R=CH3 b: R= SO2C6H5

DCC R LiAlH , ether R N 4 N reflux, 17 h, a: pyridine, 48 h, N N 82%, R= CH r.t., 59%, R= CH3 H 3 H b: acetonitrile, 18h, O r.t., 78%, R= -SO2C6H5 III.34 III.35

Figure III.8 Synthesis of 4-substituted 1,4-benzodiazocin-2-ones (III.34) and reduced analogues (III.35).

Tumor-promoting teleocidins induce growth inhibition, cell adhesion and differentiation to monocytes of human promyelocytic leukemia cells (HL-60). These teleocidins such as III.36, consist of a nine-membered indole-fused heterocycle and are known to exist in an equilibrium between two conformational states (the twist form and sofa form) in solution37. The simplified structure Indolactam V (III.37) serves as a reference compound, although it has a lower activity than teleocidine B-4. BenzolactamV8 (BL-V8-310) III.9, in which the indole ring of indolactam III.37 is replaced with a benzene ring, was synthesized in an attempt to reproduce the active conformation of teleocidins. The resulting 8-membered lactam III.9 was found to be more active than indolactam-V III.37, which was attributed to mimicking the teleocidine twist conformation in solution. The twist-restricted benzolactam-V8-310 (III.9) caused growth inhibition and differentiation of HL-60 cells at a concentration of 10 nM which is one order weaker than that of teleocidin B-4 but 30 times stronger than that of indolactam-V16, 38, 39.

Furthermore, it has been suggested that not only the 5-(S)-configuration of benzolactam-V8 III.9 40 (or the 9-(S)-configuration of indolactam-V III.37), but also the lactam hydrogen atom, play an important role for the biological activity (figure III.9) 41.

Part 1: Introduction

H 10 O H C N 3 N OH H 13 11 N 9 1 3 NH O H3C 2 4 N OH H3C 12 N 5 4 O 8 5 10 OH N 3 6 H 6 1 N 2 9 7 7 H 8

III.36 III.37 III.9

Teleocidine B-4 Indolactam-V Benzolactam-V8-310 (BL-V8-310)

Figure III.9 The structures of Teleocidine B-4 III.36, indolactam-V III.37 and the analogue Benzolactam-V8-310 III.9.

In view of this, Endo and coworkers did a lot of synthetic efforts to synthesize benzolactams-V8 with a variety of substituents42,43. The general strategy consisted of the synthesis of N-Boc- protected α,ω-aminocarboxylic acids which (exemplified by III.41, figure III.10) could be cyclized after succinimidyl ester activation to obtain the 1,4-benzodiazocin-3-one skeleton of benzolactam-V8 derivatives 16, 38, 42, 44, 45, 46. As an example, the synthesis towards diastereomeric III.9 and III.42 (figure III.1045) starts from the reaction of 4-bromomethyl-3-nitrobenzaldehyde III.38 with diethyl acetamidomalonate III.39, followed by Wittig reaction giving III.40 which was transformed to the strategic N-Boc aminocarboxylic acid III.41. Condensation with N- hydroxysuccinimide using DCC gave the activated esters (96%) which was subjected to Boc removal using CF3COOH and cyclization (carried out under dilute conditions) giving III.9 (48 %) and the epimers III.42(43 %).

Part 1: Introduction

NO2 NO 2 NHAc Br NHAc COOEt 1) NaH, DMF OHC + EtOOC COOEt H COOEt + - - 2) C9H19P Ph3 Br n-BuLi, THF, 55% III.38 III.39 C8H17 III.40

9 steps

O O

1) DCC, HOSu,CH3CN, 96% H C NH NH 3 N COOH H3C N H3C N 2) CF COOH, CH Cl 3 2 2 NHBoc OH + OH 3) Sat.NaHCO3aq, CH3COOEt (60/1) C10H21 OH C10H21 C10H21

III.9 III.42 III.41 48% 43%

Figure III.10 Illustration of the synthesis of the Benzolactams-V8 ring system according to Endo and coworkers45.

Ma Dawei and coworkers 47, 48 were able to develop an alternative synthetic route for the synthesis of benzolactam-V8 with overall yield 17.7%. The aldehyde III.44, prepared from 2-iodobenzoic acid III.43, was condensed with methyl-2-nitroacetate under the action of TiCl4 and NMM to furnish olefin III.45. A Pd/C-catalyzed hydrogenation yielded crude amino acid III.46, which was cyclized under the action of DPPA to afford a mixture of diastereoisomers III.47 and III.48.

Finally, LiBH4 reduction of III.48 afforded benzolactam-V8 III.49 (figure III.11).

Part 1: Introduction

I Me Me N COOBn MeOOCCH2NO2, N COOBn COOH TiCl4, CHO COOMe NMM, 82% NO2

III.43 III.44 III.45

Pd/C, H2, MeOH

Me N COOBn

NH2 COOMe III.46 O O P O DPPA: N3 DPPA, DMF 0.02M, 35%

O O H H N N Me N Me N COOMe + COOMe

III.47 III.48 O H N Me N

OH LiBH4, THF, 88%

III.49 benzolactam-V8

Figure III.11 Synthetic route towards benzolactam-V8 described by Ma Dawei.

Part 1: Introduction

III.5 1,5-BENZODIAZOCINES

III.5.1 Comprehensive literature review 1,5-benzodiazocine-2,6-diones

Our proposed 1.5-benzodiazocine-2,6-diones are only very limitedly described in the literature. The following paragraphs will give a comprehensive overview of reported derivatives and their synthesis.

III.5.1.1 Synthesis of Auret, Grundon and McMaster (1968)

Starting from the epoxide-ester III.51 (formed from a Darzen reaction between benzaldehyde and chloroacetanilide III.50), followed by N-methylation and treatment with an excess of methyl amine, benzodiazocinedione III.53 could be obtained (figure III.12)49. Reaction with tosyl chloride followed by elimination led to compound III.55.

O O O Cl Ph Me O N N N OH H PhCHO, NaOMe R MeNH2 O MeOH O N Ph OMe 94% OMe O Me III.50 III.51 R = H KOtBu, MeI, III.53 82% III.52 R = Me

TsCl, pyridine Me O Me O 95% N KOtBu N OTs

Ph tBuOH N N Ph O Me 100% O Me

III.55 III.54

Figure III.12 Synthesis of Auret, Grundon and McMaster

III.5.1.2 Synthesis of Gatta and Landi-Vittory (1970)

Gatta described a synthetic route for compounds III.60 (R = phenyl or benzyl), via cyclization of the open-chain precursors III.59 under the action of ethyl chloroformate (figure III.13)50. The open-chain precursors themselves were prepared from the nitro derivatives III.57, obtained from

Part 1: Introduction the reaction of 2-nitrobenzoyl chloride with the ethyl ester of N-benzyl or N-phenyl-β-alanine (III.56)51. Note that in case of direct catalytic hydrogenation of the nitroester III.57 no ring-closed product III.60 is observed (these are conditions which are known to readily give the corresponding seven membered 1,4-benzodiazepine-2,5-diones). Compounds III.60 can undergo a methylation reaction at N1 position with methyl iodide after deprotonation with sodium methoxide leading to the analogs III.61.

NO2 H NO N OEt NO2 KOH 2 R COCl O O O Ph: 74% Ph: 90% Bn: 85% N OEt Bn: 81% N OH R R O O III.56 III.57 III.58 R = Ph, Bn Ni/H Ni/H Ph: 75% 2 2 Bn: 82%

Me O O N HN NaOMe, MeI NH2 ClCOOEt, Et3N O DMF dioxane N N Ph: 54% O R Ph: 73% O R N OH Bn: 57% Bn: 63% R O III.61 III.60 III.59 R = Ph, Bn

Scheme III.13 Synthesis of 1,5-benzodiazocine-2,6-diones by Gatta and Landi-Vittory.

An alternative approach via cyclization of intermediate β-chloropropionyl anthranilamide III.62 did not furnish the desired lactam, as elimination reaction was observed (III.65) in the presence of sodium hydride or trimethylamine, whereas treatment with sodium methoxide gives rise to methyl ethers III.63 and III.64 (figure III.14).

Part 1: Introduction

O OMe

NH NaOMe N OMe + NHR N R O Cl O O III.63 III.64 NH X Ph: 54% Ph: 25% NHR Bn: 53% Bn: 28%

O III.62 O R = Ph, Bn NaH or Et3N NH NaH Et N NHR 3 81% O Ph 72% III.65 Bn 78% 82%

Figure III.14 Attempted cyclization of chlorobenzamides III.62.

We suggest that the presence of a secondary amide function is responsible for this observed behavior, as it is known that secondary amides are generally exclusively present as their Z- (trans) isomer which is geometrically unfavorable for cyclization (figure III.15).

O Cl H O NH N X NHR NH Cl R O O Z-III.62 E-III.62 (trans) (cis)

Figure III.15 Internal secondary "trans" - isomer as a possible cause for the failure of the ring closure of III.62.

III.5.1.3 Synthesis of Möhrle, Rohn and Westle (1998)

After a more recent study of the oxidation behavior of γ-carbolines, Möhrle reported that treatment of mebhydrolin (III.66, R = Bn, figure III.16) with sodium periodate afforded a number of

Part 1: Introduction different products, including benzodiazocinedion III.68.52 This procedure was also applied to N- Me and N-Ph analogues.

Me R O N COO N O NaIO4 NaIO4 H Me N N R = H N R = Me, Ph, Bn N H H R Me: 15% O Me III.67 III.66 Ph: 18% III.68 Bn: 18% Figure III.16 Access to 1.5-benzodiazocine-2,6-diones via oxidation of γ-carbolines.

The reaction mechanism of this transformation hypothesized by the authors is depicted in figure 53 III.17 . In a first step, the indole ring of III.66 can be readily oxidized at the C2-C3 bond to give the benzazonine skeleton III.69 54, 55. The present α-amino ketone functionality is not stable and reacts under oxidative conditions further via the iminium ion III.70, which is trapped with water to give the hemiaminal III.71. This is in equilibrium with the open form III.72 which can give the alternative eight-membered hemi-aminal III.7356. Hydroxide elimination and hydration of the aldehyde function give rise to the iminium salt III.75 which can undergo a further periodate oxidation to the final 1,5-benzodiazocine-2,6-dione III.68.

Part 1: Introduction

OH O Me O Me O Me NaIO 4 N NaIO4 N H2O N III.66 N O N O N O R R R III.69 III.70 III.71 R=H, Me, Ph, Bn O Me N O Me OH O HN O Me NaIO 4 HN N N O Me O R R = H N O III.68 III.67 R - -IO3 III.72 -HCOOH

O O O O O OH I I O O O Me O OH O Me Me O HO Me HO N N N H O N NaIO4 2 R = Me, N N N N Ph, Bn R O R O R O R O III.74 III.73 III.76 III.75

Figure III.17 Proposed mechanism for the periodate oxidation of γ-carbolines to 1,5- benzodiazocine-2,6-diones III.68.

It is important to note that if III.66 concerns a NH-substituted indole nucleus (R = H), the corresponding benzodiazocinedione is not formed, but rather the open derivative III.67, probably via the displayed alternative route.

O O O O X NHMe O NHMe N N H H O Z-III.72 E-III.72 (trans) ( cis)

Figure III.18 Internal secondary "trans" - amide in III.72 (R=H) as a possible cause for not forming hemiaminal III.73 (R=H).

Part 1: Introduction

Again, we may suggest that this is a consequence of the presence of an internal secondary amide function having the trans geometry, so that ring contraction on the stage of III.72 (on the way to III.73) cannot occur, and alternative periodate oxidation to the carboxylic acid III.67 takes place (figure III.18).

III.5.1.4 Synthesis of Karp and colleagues (1998)

Synthesis of a single 1,5-benzodiazocine-2,6-dione III.81 was reported by Karp and coworkers57. Reaction between the methyl ester of N-tert-butyl β-alanine III.77 and 5-chloro-2-nitrobenzoyl chloride, followed by hydrogenation provided amino-ester III.79 which underwent hydrolysis followed by cyclization using DCC to give the desired 1,5-benzodiazocinedione III.81 (figure III.19), which was tested for herbicidal activity, but was found to be inactive.

NO2 NO2 O O Cl Cl H H , Pd/C N OMe Cl 2 N OMe O Et3N, THF EtOAC O III.77 III.78

HN NH2 NH2 DCC NaOH O O Cl N CH CN Cl Cl 3 H2O/THF O N OH N OMe

O O III.81 III.80 III.79

Figure III.19 Synthesis of Karp via cyclization of α,ω-aminocarboxylic acid III.80.

III.5.2 1,5-benzodiazocine-2-one via imine formation

Derieg described the synthesis of 8-chloro-3,4-dihydro-1-methyl-6-phenyl-1,5-benzodiazocine-2- one III.11 (the direct ring homologue of diazepam) via cyclization of the open-chain precursors III.84 under reflux in toluene (figure III.20)18.The open-chain precursor is prepared from treatment of carboxybenzyl-β-alanine III.82 with PCl5 leading to the acid chloride which was

Part 1: Introduction treated with 5-chloro-2-methylamino benzophenone to give III.83. The acid hydrolysis of III.83 provides the amine III.84.

O N NHCbz 1) Ether, 0°C, PCl5, 1.5 h HBr (31%), CH COOH,2 h, r.t HO NHCbz O 3 2) 5-chloro-2-methylamino Cl O NH4OH benzophenone, CHCl3, r.t. 3) water, 3N NaOH, 3N HCl

III.82 III.83

CH 3 O N NH 2 N Toluene, reflux o.n. O Cl O 23.4% Cl N

III.84 III.11

Figure III.20 Synthesis of 1,5-benzodiazocine-2-one III.11.

III.6 1,6-BENZODIAZOCINES

III.6.1 Via Intramolecular nucleophilic substitution (Stetter)

The first report on the 1,6-benzodiazocine58 skeleton described the condensation of o-phenylene diamine sulfonamide III.85 with dibromobutane III.86 to produce N,N-ditosyl-1,6- benzodiazocine III.87, which was treated with H2SO4 to liberate the amino groups to afford the parent 1,6-benzodiazocine III.88 (figure III.21).

Ts Ts HN NH N Na-BuOH H2SO4 + Br(CH2)4Br 35% 70% NH N N H Ts Ts III.85 III.86 III.87 III.88

Figure III.21 Synthesis of 1,2,3,4,5,6-hexahydro[b][1,4]diazocine III.88.

Part 1: Introduction

III.6.2 Via Schmidt rearrangement (Kawamoto)

An attempt to enlarge the benzazepinone ring in III.89 by one nitrogen atom using the Schmidt reaction unexpectedly afforded benzimidazole derivative III.94 as the major component, along with a small amount of the eight-membered lactams III.92 and III.93 via intermediates III.90, III.91, figure III.2259.

H N C+ N HN H HN N N III.90 CCl3COOH H2O + + N NaN N N 3 H H O O O H III.92 III.93 III.94 III.89 N minor minor major (42%) C N

III.91 Figure III.22 Attempted synthesis of eight-membered lactam III.92 using the Schmidt rearrangement.

III.6.3 Via Lactamization (Elguero).

The strategy of Elguero and coworkers60 involves a base-catalyzed condensation of o- phenylenediamines III.95 and diethyl succinates III.96 to obtain a series of five 1,6- benzodiazocine-2,5-dione derivatives (III.97 - III.101) (figure III.23). Interestingly, the presence of an internal secondary amine does not prevent the successful cyclization to the eight-membered ring system.

Part 1: Introduction

R1 O R1 O O N NH NaH, THF R3 + R NH 3 N O R2 O R2 O

III.97 R1=R2=R3=H, 40% III.98 R1=R2=CH3, R3=H, 10% III.95 III.96 III.99 R1=CH3, R3=R2=H, 10% III.100 R1=R2=H, R3=CH3, 45% III.101 R1=R2=R3=CH3, 35%

Figure III.23 Synthesis of a series of 1,6-benzodiazocine-2,5-dione derivatives via condensation of o-phenylenediamines and succinates.

III.6.4 Via intramolecular N-acylation (Yang et al.).

Coupling of N-phosphoamino acid III.103 with 2-bromoaniline III.102 in pyridine using 2 equiv of diphenyl phosphite (DPP)gave amide III.104 which was cyclized via a Cu-catalyzed reaction to give 1,6-benzodiazocine-2-ones III.105. Deprotection of in acidic medium led to compound III.92 (figure III.24).61

O H H O N NH 5 mol % CuI N 2 HOOC DPP, Py 10 mol % proline + HN 65-95% 2 eq K CO Br Br 2 3 N DIPP toluene, 110 °C NH DIPP DIPP 72 h, 68% III.102 III.103 III.104 III.105

H O N acid O DIPP: O P O N H DIPP-: diisopropylphosphoryl III.92

Figure III.24 Synthesis of 3,4,5,6-tetrahydrobenzo[b][1,4]diazocin-2(1H)-one III.92, via Cu- catalyzed intramolecular N-arylation (DIPP= diisopropylphosphoryl)

Part 1: Introduction

Compared to N-protected intermediate III.104, the corresponding intermediate containing a free primary amino group product failed to cyclize by the same conditions. The authors hypothesize that introduction of phosphoryl at the N-terminus could conformationally restrain the acyclic precursor, which could be favorable for intramolecular reaction. According to the authors, a simplified mechanistic view for the formation of cyclic products III.105 from the open forms III.104 is summarized in figure III.25.

O O H H O H N N N or CuL Br O Br Br NH N = CuL NH LCu NH DIPP H O DIPP DIPP Cu III.104 III.107 III.108 III.106

O O H O H N HN N or CuL N N N Cu DIPP DIPP DIPP L III.105 III.109 III.110

Figure III.25 Possible copper-catalyzed cyclization mechanism61.

The copper catalyst III.106 resulting from the reaction of cuprous ion with proline salt could undergo coordination with the two nitrogen atoms of the amide and the DIPP-NH leading to the complex III.107, which places both bromine and DIPP-NH in the appropiate orientation for formation of III.109. Alternatively the coordination of copper catalyst III.106 with the phenyl ring and nitrogen of the DIPP-NH could afford the Л-complex III.108, which can also undergo the intramolecular N-arylation providing the desired nitrogen heterocycle III.110. Decomplexation of copper catalyst system in III.109 or III.110 yields the target product III.105.

Part 1: Introduction

III.6.5 Via reductive ring expansion (Shvidenko et al.)

The reduction of benzimidazole derivative III.94 in toluene using a six-fold excess of DIBAL-H led to the formation of a mixture of 1,6-benzodiazocine III.88 and o-phenylenediamine derivative III.111 (figure III.26)62.

DIBAL-H N N N HN NH + NH2

III.94 III.88 III.111 35% 43%

Figure III.26 Synthesis of 1,2,3,4,5,6-hexahydrobenzo[b][1,4]diazocine by Shvidenko.

The formation of compounds III.88 can be easily rationalized by the reduction mechanism of amidines with DIBAL-H which includes a sequential electrophilic attack of DIBAL-H at the two nitrogen atoms ((figure III.27). path 1). At the same time the reductive cleavage of the C-N bond resulting in intermediates III.112 may be implemented by a double attack at the same nitrogen atom to form aluminum imides III.114 to afford the compound III.111 ((figure III.27). path 2), which are usually less energetically favorable compared to intermediates III.11362.

i-Bu2Al N

N III.88 " H " 1 Al(i-Bu)2 Path 1 H Al-H " H " Al(i-Bu) DIB N N N 2 N 2 III.113

DIBAL-H D IB Al Pa -H th N III.111 III.112 2 III.94 N(Ali-Bu2)2

CH3 H CH3 DIBAL-H: Al H3C CH3 III.114

Figure III.27 Assumed mechanistic paths for the DIBAL-H reduction of III.94.

Part 1: Introduction

III.6.6 Via ring-closing metathesis (Taher, Van Otterlo)

A set of 1,6-benzodiazocines was synthesized by ring-closing metathesis63,64 from the corresponding bis-allyl o-phenylenediamine precursors using Grubbs second generation catalyst. (figure III.28). Ru-catalyzed isomerization was shown to give isomeric products III.119 and III.119`.

R R R NH N Grubbs second N NaH or K2CO3 generation catalyst + Br NH N III.118a R: Boc: 49% N III.117a R: Boc : 66% R R III.117b R: Bz : 81% III.118b R: Bz : 97% R III.118c R: Ts : 92% III.117c R: Ts : 92% III.117 III.118 III.115 III.116 III.117d R: Ac : 92% III.118d R: Ac : 80%

R R CH3 N N H3C [RuClH(CO)(PPh3)3] N N and/or CH N N H C 3 3 CH H3C R R 3 III.119 III.119` Cl Ru Cl Ph III.119a R: Boc III.119b R: Bz PCy3 III.119c R: Ts III.119d R: Ac Grubbs Catalyst, 2nd Generation

Figure III.28 Synthesis of a series of 1,6-benzodiazocines using Grubbs second generation RCM catalyst.

III.6.7 Via thermal ring expansion (Shue)

According to Shue and Fowler, N,N-dimethylbenzo-1,6-dihydro-1,6-diazocine III.12365 was synthesized through the thermal ring opening of cyclobutene III.122. Gas phase thermolysis at 285 °C produces the benzodiazocine III.123 as the only detectable product. Irradiation of diazocine III.123 results in the reversal formation of cyclobutene III.122 (figure III.29).

Part 1: Introduction

Me Me Me Cl N N NH n-BuLi  + -78 C N NH Cl h N Me Me Me

III.120 III.121 III.122 III.123

Figure III.29 Synthesis of 1,6-benzodiazocine III.123 via gas phase thermolysis of III.122.

III.7 2,3-BENZODIAZOCINES

III.7.1 Via cycloaddition (Dennis)

The only known literature example66 describes the reaction of betaine III.124 with phenylacetylene III.125 in refluxing xylene to give two isomeric products. Besides the normally expected cycloadduct III.126 the 2,3-benzodiazocine III.127 was isolated. (figure III.30).

O- Ph N O O Ph N Ph N N Xylene, reflux N N+ + Ph + Ph

III.124 III.125 III.126 III.127 minor major 10% 75%

Figure III.30 Synthesis of the 2,3-benzodiazocine III.127.

From the authors point of view the diazocine III.127 was obtained from the normal cycloadduct III.126 via electrocyclic ring opening reaction as illustrated in (figure III.31).

Part 1: Introduction

Ph N O Ph N Ph N O Ph O Ph N N N III.128 Ph

III.126 III.127

Figure III.31 Proposed mechanism for the formation of 2,3-benzodiazocine III.127.

III.8 2,4-BENZODIAZOCINES

III.8.1 Via intramolecular aminal formation

Starting from 4-isoquinolinecarboxylic acid III.129, Kametani and coworkers67 synthesized bridged 2,4-benzodiazocines III.133 and III.134 via consecutive amide formation, catalytic hydrogenation of the heteoaryl moiety and LiAlH4 reduction to bisamine III.132, which was subjected to aminal formation using appropriate aldehydes to give the cyclized targets III.133 and III.134 (figure III.32).

Part 1: Introduction

CONHCH C H COOH CONHCH2C6H5 2 6 5

PhCH2NH2 H2, PtO2 N NH N POCl3, 88% 80%

III.129 III.130 III.131

LiAlH4, 84%

CH2NHCH2C6H5

N CH2O, benzene NH N Reflux, 81%

III.133 III.132

PhCHO, MeOH, 66%

N C6H5

III.134

Figure III.32 Synthesis of bridged 2,4-benzodiazocines.

III.9 2,5-BENZODIAZOCINES

III.9.1 Via nucleophilic substitution

Reaction of 1,2-bis-bromomethyl-3,4,5,6-tetrachloro-benzene III.135 with 2- dimethylaminoethylamine III.136 afforded the quaternary ammonium salt 2,2-dimethyl- 1,2,3,4,5,6-hexahydro-7,8,9,10-tetrachloro-2,5-benzodiazocinium bromide68 (III.137) (figure III.33).

Part 1: Introduction

Cl Me Me Cl Cl N Me Br Me N + Cl Br- Br Cl H2N Cl Cl N Cl H

III.135 III.136 III.137

Figure III.33 Synthesis of 2,5-benzodiazocine III.137.

Similarly, Sawanish and coworkers reported the synthesis of compound III.14069 through condensation of III.139 and α,α′-dibromo-o-xylene III.138 in basic medium (figure III.34). The compound was shown to suppress the efflux of vinblastine from P388/ADR cells and increased its intracellular accumulation, while it barely increased the vinblastine accumulation in sensitive cells (P388/S).

Ts Ts NH N Br Na, n-BuOH + Br HN N Ts Ts

III.138 III.139 III.140

Figure III.34 2,5-benzodiazocine synthesized by Sawanish and coworkers.

The copper catalyzed reaction of bis acetate III.141 with N,N-diphenylethane-1,2-diamine III.142 using Ph-PyBOX as a ligand afforded compound III.14370 in 68% yield as a mixture of two diastereoisomers (meso-isomer/DL-isomer = 8/1), the minor DL-isomer was obtained with 66% ee. (figure III.35).

Part 1: Introduction

Ph PhHN N 5 mol%, CuOTf.1/2C6H6 OAc 10mol% Ph-PyBOX OAc + NHPh 2.4 equiv i-Pr2NEt N MeOH, r.t., 20 h Ph

III.141 III.142 III.143 O O N 68% yield (meso/dl=8/1), 66% ee N N Ph Ph-PyBOX Ph

Figure III.35 Synthesis of benzodiazocine III.143.

III.9.2 Via Ugi/carbonylative lactonization sequence

Various amines III.144, ortho-iodobenzaldehydes III.145, isocyanides III.146 and Boc- aminoacids III.147 were reacted in an Ugi 4-component reaction and the products purified via sequential treatment with PS-trisamine and MPCO3 resins to afford pure Ugi products III.148 (figure III.36) in respectable yields (70 - 88%) as mixtures of diastereomers71, where applicable.

R3 NHBoc O R1 O R3 R2 NC R N O N 1 R III.146 2 NH I O 1. 4N HCl HN + Ugi MCR NH R R1 NH2 H 2 + + MeOH 2. Mo(CO)6, O III.144 R3 O I Pd(OAc)2 HN COOH III.145 III.148 III.149 Boc (70-88%) (33-77%) III.147 Figure III.36 Library of 2,5-benzodiazocines via the Ugi- carbonylative lactamization sequence.

Deprotection of the Ugi MCR products with 4N HCl was followed by a carbonylation/ intramolecular amidation reaction, delivering the intended sturucturally diverse 2,5- benzodiazocine-3,6-dione III.149 library in a moderate yields (33-77 %) (figure III.36).

Part 1: Introduction

O O NH O O O N N O NH NH HN O O O

III.150 III.151

Figure III.37 Examples of compounds synthesized by Vasudevan and coworkers.

III.10 3,4-BENZODIAZOCINES

III.10.1 Via hydrazone formation Dicarboxylic acid III.152 was converted to diketones III.153 which were cyclized using hydrazine hydrobromide to afford 3,4-benzodiazocines III.154 and III.155 (figure III.38)72.

R O R NH2NH2.HBr N COOH 1. SOCl2 N COOH 2. Ar-H, AlCl3 R O R

III.152 III.153 III.154 R= phenyl III.155 R= 2,4-xylyl

Figure III.38 The 3,4 benzodiazocines synthesized by Norman.

Part 1: Introduction

References:

1 Alvarez, M.; Joule, J. A. Alkaloids (Academic Press), 2001, 57 (Chemistry and Biology), 235-272. 2 Bertha, C. M.; Ellis, M.; Flippen-Anderson, J. L.; Porreca, F.; Rothman, R. B.; Davis, P.; Xu, H.; Becketts, K.; Rice, K. C. J. Med. Chem. 1996, 39, 2081-2086. 3 Iliopoulou, D.; Vagias, C.; Harvala, C.; Roussis, V. Phytochemistry 2002, 59, 111-116. 4 Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Advanced Drug Delivery Reviews 2001, 46, 3 -26 5 Harrison, R. J.; Gowan, S. M.; Kelland, L. R.; Needle, S. Bioorg. Med. Chem. Lett. 1999, 9, 2463-2468. 6 Watthey, J. W. H.; Gavin, T.; Desai, M. J. Med. Chem. 1984, 27, 816-818. 7 Sicher, J. Prog. Stereochem. 1962, 3, 202-263. 8 Patil, A. D.; Freyer, A. J.; Killmer, L.; Carte, P. B.; Jurewicz, A. J.; Johnson, R. K. Tetrahedron 1997, 53, 5047 –5060. 9 Popović, V.; Stojković, D.; Nikolić, M.; Heyerick, A.; Petrović, S.; Soković, M. and Niketić, M. Food Funct 2015, 6, 1205-1211. 10 Sternbach, L.; Reeder, E.; Keller O.; Metlesics, W. J. Org. Chem. 1961, 26, 4488-4497. 11 Nicholls, I. A.; Alewood, P. F.; Brinkworth, R.I.; Morrison, S.F. and Andrews, P. R. J. Cherm. Research 1993, 5(1), 408-409. 12 Ott, H. U.S. Patent, 3577557 (1971); (Chem. Abstr. 1971, 75, 49158v). 13 Yamamoto, H.; Inaba, S.; Okamoto, T.; Hirohashi, T.; Ishizumi, K.; Yamamoto, M.; Maruyama I.; Mori, K.; Kobayashi, T.; Japanese Patent, 71 04,177 (1971): (Chem.Abstr. 1971, 74, 141912p). 14 Koch, U.; Narjes, F.; Curr. Topics Med. Chem. 2007, 7, 1302–1329. 15 a) Herold, P.; Herzig, J. W.; Wenk, P.; Leutert, T.; Zbinden, P.; Fuhrer, W.; Stutz, S.; Schenker, K.; Meier, M.; Rihs, G. J. Med. Chem. 1995, 38, 2946-2954; b) Zimmermann, N.; Bokník, P.; Gams, E.; Herzig, J. W.; Neumann, J.; Schmitz, W.; Scholz, H.; Wenzlaff, H. J. Pharmacol.Exp. Ther. 1996, 277, 1572-1578. 16 Endo, Y.; Yamagughi, M.; Hirano, M.; Shudo, K. Chem. Pharm. Bull. 1996, 44(5), 1138-1140. 17 Dawei, M.; Guozhi T.; Kozikowski A. P.; Org. let. 2002, 4(14), 2377-2380. 18 a) Yamamoto, H .; Inaba, S .; Okamoto, T .; Hirohashi, T .; Ishizumi, K .; Yamamoto, M .; Maruyama, I .; Mori, K .; Kobayashi, T. US Patent 3697508, (1972). b) Derieg, M.E.; Schwein Inger, R. M.; Fryer, R. I. J. Org. Chem. 1969, 34, 179-183. c) Denzer, M.; Ott, H. J. Org. Chem. 1969, 34, 183-187. 19 Duncan, G. W.; Lyster, S. C.; Wright, J. B. Proc. Soc. Exp. Biol. Med. 1965, 120,725-728. 20 Nonnenmacher, E.; Hever, A.; Mahamoud, A.; Aubert, C.; Molnar, J.; Barbe, J. Org. Prep. Proced. Int. 1997, 29, 711-714. 21 Fink, B. E.; Gavai, A. V.; Tokarski, J. S.; Goyal, B.; Misra, R.; Xiao, H.-Y.; Kimball, S. D.; Han, W. C.; Norris, D.; Spires, T. E.; You, D.; Gottardis, M. M.; Lorenzi, M. V.; Vite, G. D. Bioorg. Med. Chem. Lett. 2006, 16, 1532-1536. 22 Wakankar, D. M.; Hosangadi, B. D. Indian J. Chem. 1984, 23B, 136-139. 23 Marsella, M. J. Acc. Chem. Res. 2002, 35, 944-951. 24 Suga, T.; Wi, S.; Long, T. E. Macromolecules 2009, 42, 1526-1532. 25 a) Sergeyev, S. Helv. Chim. Acta 2009, 92, 415-444. b) Dolensky, B.; Elguero, J.; Kral, V.; Pardo, C.; Valik, M. Advances in Heterocyclic Chemistry; AP: New York, 2007; Vol. 93, pp 1-56. C) Adrian, J. C.; Wilcox, C. S. J. Am. Chem. Soc. 1989, 111, 8055-8057. 26 a) Baldeyrou, B.; Tardy, C.; Bailly, C.; Colson, P.; Houssier, C.; Charmantray, F.; Demeunynck, M.

Part 1: Introduction

Eur. J. Med. Chem. 2002, 37, 315-322. B) Bailly, C.; Laine, W.;Demeunynck, M.; Lhomme, J. Biochem. Biophys. Res. Commun. 2000, 273,681-685. c) Johnson, R. A.; Gorman, R. J.; Wnuk, N. J.; Crittenden, N. J.; Aiken, J.W. J. Med. Chem. 1993, 36, 3202-3206. 27 (a) Illuminati, G.; Mandolini, L. Acc. Chem. Res. 1981, 14, 95-102; (b) Evans, P. A.; Holmes, A. B. Tetrahedron 1991, 47, 9131-1966; (c) Harmata, M.; Ying, W.; Barnes, C. L. Tetrahedron Lett. 2009, 50, 2326-2328; (d) Lu, S. M.; Alper, H. J. Am. Chem. Soc. 2008, 130, 6451-6455; (e) Maier, M. E. Angew. Chem., Int. Ed. 2000, 39, 2073-2077; (f) Ferrer, C.; Echavarren, A. M. Angew. Chem., Int. Ed. 2006, 45, 1105-1109; (g) Petasis, N. A.; Patane, M. A. Tetrahedron 1992, 48, 5757-5821; (h) Mehta, G.; Singh, V. Chem. Rev. 1999, 99, 881-930; (i) Yet, L. Chem. Rev. 2000, 100, 2963-3008; (j) Ma, S.; Gu, Z. J. Am. Chem. Soc. 2006, 128, 4942-4943. 28 Grasso, S.; Zappala, M.; Chimirri, A. Heterocycles, 1987, 26(9), 2477-2501. 29 Hanno, S.; Christian, N.; Rainer, H. Beilstein Journal of Organic Chemistry 2013, 9(1), 1-7. 30 Merino, E.; Ribagorda, M. Beilstein J. Org. Chem. 2012, 8, 1071–1090. 31 Kempter, G. M.; Mezhdu Nas. Imp. Stran-Chlenov SEV. 10th, 1975, 1, 146. (Chem. Abstr., 1978, 89, 6313p). 32 Kempter, G., Ziegner, H. J.; Moser, G.; Natho W.; Wiss. Z. Paedagog. Hochsch. 1977, 21, 5. (Chem. Abstr. 1978, 89, 163560). 33 Ian A. Nicholls, Paul F. Alewood, Ross I. Brinkworth, Stuart F. Morrison and Peter R. Andrews J. Chem. Research (S), 1993, 1, 408-409. 34 Kempter,G.; Mengs, H. J.; Spindler, J.; Kuehne, S. Ger. (East) Pat. DD235.452 (1986). 35 Muchowski, J. M. Can. J. Chem. 1970, 48, 1946-1948. 36 Muchowski, J. M. Can. J. Chem. 1971, 49, 2023-2028. 37 (a) Endo, Y.; Hasegawa, M.; Itai, A.; Shudo, K.; Tori, M.; Asakawa, Y.; Sakai, S. Tetrahedron Lett. 1985, 26, 1069-1072. (b) Endo, Y.; Shudo, K.; Itai, A.; Hasegawa, M.; Sakai, S. Tetrahedron 1986, 42, 5905-5924. 38 Endo, Y.; Ohno, M.; Takehana, S.; Driedger, P.E., Stabel, S.; Shudo. Chem. Pharm. Bull. 1997, 45(2), 424-426. 39 Okabe, S.; Sueoka, N.; Komori, A.; Suganuma, M.; Endo, Y.; Shudo, K.; Fujiki. H. Biol Pharm Bull. 1998, 21(5), 465-468.The same amounts (5.5 nmol/application) of (-)-BL-V8-310 and teleocidin B- 4 induced tumors on mouse skin initiated with 7,12-dimethylbenz[a]-anthracene (DMBA) in 13.3% and 86.7%, respectively, of tumorbearing mice in week 20. 40 Endo, Y.; Ohno, M.; Hirano, M.; Takeda, M.; Itai, A.; Shudo, K. Bioorg. Med. Chem. Lett. 1994, 4, 491-494. 41 Nakagawa, Y.; Irie, K.; Nakamura, Y.; Ohigashi, H. Bioorg. Med. Chem. Lett. 2001, 11, 723-728. 42 (a) Endo, Y.; Shudo, K.; Okamoto Chem. Pharm. Bull. 1982, 30, 3457-3460. (b) Endo, Y.; Shudo, K.; Furuhata, K.; Ogura, H.;Sakai, S.; Aimi, N.; Hitotsuyanagi, Y.; Koyama, Y. Chem. Pharm. Bull. 1984, 32, 358- 361. 43 Fujiki, H.; Suganuma, M.; Hakii, H.; Nakayasu, M.; Endo, Y.; Shudo, K.; Irie, K.; Koshimizu, K.; Sugimura, T. Proc. Jpn. Acad., Ser. B 1985, 61, 45-47. 44 Endo, Y.; Yokoyama A. Bioorg. Med. Chem. Lett. 2000, 10, 63-66. 45 Ohno, M., Endo, Y., Hirano, M., Itai, A.; Shudo K. Tetrahedron letters 1993, 34(50). 8119-8122. 46 Endo,Y.; Takehana, S.; Ohno, M.; Driedger, P. E.; Stabel, S.; Mizutani, M. Y.; Tomioka, N.; Itai, A.; Shudo, K. J. Med. Chem. 1998, 41, 1476-1496. 47 Dawei, M.; Zhang, Y.; Yao, J.; Wu, S.; Tao, F. J. Am. Chem. Soc. 1998, 120, 12459-12467.

Part 1: Introduction

48 Dawei, M.; Tang, W. Tetrahedron letters 1998, 39, 7369-7372. 49 Auret, B.J.; Grundon, M.F.; McMaster, I.T. J. Chem. Soc. C. 1968, 2862-2863. 50 Gatta, F.; Landi-Vittory, R. Farmaco, Ed. Sci. 1970, 25, 991-998. 51 Stork, G.; Mc Elvan, S.M. J. Am. Chem. Soc. 1947, 69, 971-972. 52 Möhrle, H.; Nestle, W.; Westle, G. Pharmazie 1998, 60, 741-748. 53 Möhrle, H.; Rohn, C.; Westle, G. Pharmazie 2006, 61, 391-399. 54 Dolby, L.J.; Booth, D.L. J. Am. Chem. Soc. 1966, 88, 1049-1051. 55 Dolby, L.J.; Rodia, R.M. J. Org. Chem. 1970, 35, 1493-1496. 56 Möhrle, H.; Haug, W.; Federolf, E. Archiv der Pharmazie 1973, 306(1), 44-54. 57 Karp, G.M.; Manfredi, M.C.; Guaciaro, M.A.; Harrington, P.M.; Marc, P.; Ortlip, C.L.; Quakenbush, L.S.; Birk, I.T. “1H-1,4-Benzodiazepine-2,5-diones and Related Systems: Synthesis and Herbicidal Activity” in “Synthesis and Chemistry of Agrochemicals V”; Baker, D.R.; Fenyes, J.G.; Basarab, G.S.; Hunt, D.A. (Eds.); ACS Symposium Series 686; American Chemical Society: Washington, D.C.; 1998; pp 95-106. 58 Stetter, H. Chem. Ber. 1953, 86, 197-205. 59 Kawamoto, H.; Matsuo, T.; Morosawa, S. Bull. Chem. Soc. Jpn. 1973, 46, 3898-3899. 60 Elguero, J.; Fruchier, L.; Llouquet, G.; Marzin Can. J. Chem., 1976, 54(7), 1135-1138. 61 Yang, T., Lin, C., Fu, H., Jiang, Y., Zhao, Y. Org. Lett. 2005, 7(21), 4781-4784. 62 Shvidenko, T.; Nazarenko, K.; Shvidenko, K.; Kostyuk, A. Tetrahedron Let. 2014, 55(1), 279–281. 63 Taher, A.; Aderibigbe, B. A.; Morgans, G. L.; Madeley, L. G.; Khanye, S. D.; Westhuizen, L. v.d.; Fernandes, M. A.; Smith, V.J.; Michael, J. P.; Green, I. R.; van Otterlo, W. A.L. Tetrahedron 2013, 69, 2038-2047. 64 van Otterlo, W. A. L.; Morgans, G. L.; Khanye, S.D.; Aderibigbe, B. A. A.; Michael, J. P.; Billing, D. G. Tetrahedron Lett. 2004, 45, 9171–9175. 65 Shue, H. J.; Fowler, F. W. Tetrahydron Lett. 1971, 26, 2437-2440. 66 Dennis, N.; Katritzky, A. R.; Ramaiah, M. J. Chem. Soc. Perkin I 1976, 2281-2284 67 Kametani, T.; Kigasawa, K.; Hayasaka T. Chem. Pharm. Bull. 1965, 13(10), 1225-1230. 68 Rosen, W. E.; Toohey, V.P.; Shabica, A.C. J. Am. Chem. Soc. 1958, 80, 935-939. 69 Sawanish, H.; Wakusawa, S.; Murakami, R.; Miyamoto, K.; Tanaka, K.; Yoshifuji, S. Chem. Pharm. Bull. 1994, 42(7), 1459-1462. 70 Shibata, M.; Nakajima, K.; Nishibayashi, Y. Chem. Commun., 2014, 50, 7874-7877. 71 Vasudevan, A.; Verzal, M. K. Tetrahedron Lett. 2005, 46, 1697–1701. 72 Norman, L. A.; Gilbert, A. Y. J. Org. Chem. 1960, 25, 1509-15011.

PART 2: RESULTS AND DISCUSSION

Part 2: Results and discussion

IV. AIM AND STRATEGY IV.1 GENERAL AIM

As part of our ongoing research on medium ring heterocycles as potential new privileged structures, our interest in this project is in synthesizing 1,5-benzodiazocine-2,6-dione derivatives which have only been limitedly studied to date, although they can be considered as pharmaceutically interesting, as they are closely related to the benzodiazepine privileged structure (chapter I).

The aim of this research project is to synthesize libraries of structurally diverse compounds on the basis of a 1,5-benzodiazocine-2,6-dione skeleton, on which up to ten substitutable attachment points are in principle available (IV.1, figure IV.1). For reasons of clarity throughout this dissertation a simplified general representation (IV.2) is used where epimeric side chain R2a and

R2b are represented by a general substituent R2. Similarly R3a and R3b are represented by R3 while

R5a-R5d are represented by R5. Starting from commercially available and/or easy to synthesize building blocks the purpose will be to build up the eight-membered (bis)lactam ring using a solid- phase synthesis approach which would allow the introduction of side chain diversity during the synthesis. In this way, ideally a general synthesis methodology could be applied to the parallel construction of a combinatorial collection of 1,5-benzodiazocine-2,6-dione derivatives.

R R1 O 5d R1 O N R2a 10 R5c N R 10c 2 R 2b 9 1 3 2 R R5 4 3a 8 6 5 R5b N 6a R3 R3b 7 N R5a O R 4 O R4 IV.1 IV.2

Figure IV.1 Representative structure for the 1,5-benzodiazocine-2,6-dione skeleton

IV.2 SYNTHESIS STRATEGIES

A. General retro synthesis

The most obvious strategic disconnections in the intended 1,5-benzodiazocine-2,6-dione skeleton can be made at both lactam functionalities, (figure IV.2). The resulting synthons IV.3 and IV.4 can then be related to two possible basic building blocks: anthranilic acid

Part 2: Results and discussion derivatives IV.5 and β-amino acids IV.6. In principle, sets of these building blocks are readily available, either via commercial sources or established synthetic methodology.

R1 O R O N amide bond 1 R2 formation N R R5 2 R5 N R3 R3 O R4 N O R4 IV.2 IV.3 IV.4

R1 O NH HO R2 R5 OH HN R3 O R4 IV.5 IV.6 Anthranilic acids -amino acids

Figure IV.2 Retrosynthetic scheme towards 1,5-benzodiazocine-2,6-dione derivatives; anthranilic acids and β-amino acids as the necessary key building blocks

Our combinatorial approach is based on the use of a solid phase synthesis sequence. As such, an appropriate anchor point has to be chosen for the synthesis route on the solid support. In the solid phase synthesis of (hetero) cyclic systems where a ring closure is the key step of the synthesis, two approaches can be distinguished. In the first approach, the ring closure is accompanied with simultaneous cleavage of the final product from the solid support, and the resin can thus be considered as a leaving group ("cyclization/release strategy ", route A, figure IV.3). In the alternative strategy, cyclization led to a ring closure product that is still resin bonded ("on-resin cyclization", route B, figure IV.3). Both approaches have intrinsic advantages and disadvantages which are briefly outlined in the following paragraph.

Part 2: Results and discussion

O R2 O NH2 O R2 O NH2 RO N O N R1 R1 R4 R3 R3 IV.7 IV.8

Route A Route B cyclization/release On-resin cyclization

O H H O N R N 1 cleavage from resin R1 R3 R3 R N 2 N R2

O O R4 IV.9 IV.10

Figure IV.3 Two possible solid phase synthesis approaches: route A through simultaneous ring closure and resin cleavage; Route B where ring closure leads to resin-bound product IV.9 in which an additional cleavage step is necessary.

B. Cyclization/release vs. on-resin cyclization

The cyclization/release strategy is part of the so-called traceless linker methods, in which there is no residual functional group (trace) left on the product after cleavage1. An advantage of this strategy is that only ring-closed products are expected to be selectively cleaved from the resin, resulting in a crude product with a relatively higher degree of purity. A disadvantage is that all on- resin scaffold decoration needs to happen before ring closure. Most literature examples of this strategy make use of a nitrogen atom as the nucleophile and an ester or amide moiety as solid phase anchor functionality, resulting in the formation of five-2, six-3 or seven-membered lactams4. The success of this approach not only depends on the nucleophilicity and electrophilicity of both the reaction partners, but also on steric factors.

On the other hand, in the on-resin cyclization strategy, the ring closed products remain anchored onto the solid support. The main advantages of this approach are the possibility to derivatize the cyclized product further on-resin and to activate both reaction partners involved during the ring

Part 2: Results and discussion closure (in contrast to the above-mentioned cyclization/release approach). Main disadvantages are the loss of one diversity position (the anchoring point) and the usually lower purity of the final products compared to the cyclization-release approach, because cleavage is not selective for the ring-closed products. Based on the above-mentioned advantages and on previous successful results in our laboratory5, we decided in this research work to use the cyclization release strategy.

Applied to our target compounds, this leads to a general retrosynthetic scheme as depicted in figure IV.4. The 1,5-benzodiazocine-2,6-dione IV.2 could be obtained by cyclization through the ester bond resin-bound dipeptide precursors IV.11 which could be built up in turn from the coupling of anthranilic acid with resin-bound β-amino acid derivatives IV.12, (route A) or by changing the sequence of the building blocks (route B); both indicated routes (A and B) could essentially lead to the same benzodiazocinedion. However, prior research in our laboratory5 has shown that the amide formation between the amine group of anthranilic acid and the carboxylic group of β-amino acid (route B) provides low yields, probably because of the low nucleophilicity of that amine group as it can be considered to be a vinylogous amide.

R1 R4 O NH R1 A O O NH route A N route B R R R1 O 4 N 2 O N R R 3 5 R lactam R lactam 2 O R3 N 3 O R5 formation formation R4 O R4 B R5 IV.11 IV.2 IV.14

amide formation amide formation

R R1 O 4 NH O NH esterfication esterfication O O R3 OH R4 R5 IV.12 IV.13 IV.15 Figure IV.4 Retro synthetic approaches for a cyclization/release strategy.

Part 2: Results and discussion

C. Cyclization/release strategy: Previous work in our laboratory

C.1 The effect of secondary or tertiary amide bond [N-5 substitution]

Wang resin and HMBA-AMPS resin were used in a solid phase synthesis sequence in our laboratory to synthesize IV.19, through the resin-bound precursor IV.18. Cyclization was tested under many conditions using acid, base or nucleophilic catalysis, but all attempts failed to afford the cyclized IV.19.

X H O O O NH2 N

OH O N H 5 N H Wang resin IV.16 O or IV.18 IV.19 HMBA-AMPS IV.17 O

OH N H O OH

Wang resin HMBA-AMPS resin IV.16 IV.17

Figure IV.5 Previous attempts to cyclize secondary amide resin-bound precursor IV.18 to IV.19.

These negative results are thought to arise from the presence of the internal secondary amide. In order to obtain appropriate cyclization, the reactive centers should be able to approach each other, meaning that the amine function has to approach the ester group to carry out the desired acyl substitution. However, the internal secondary amide function affects the flexibility of the molecule; as a result of the partial double bond character of the C-N bond (IV.22), there are two possible isomers, cis (E-)isomer and trans (Z-)isomer as shown in figure IV.6.

Part 2: Results and discussion

O O O R' R' R' R N R N R N R" R" R"

IV.20 IV.21 IV.22

O O R'' R' R N R N CIP priority R' R'' R' R'' O R E-(cis) isomer Z-(trans) isomer IV.23 IV.24

Figure IV.6 E- and Z- isomers of the amide.

The population of these conformations and their isomerization kinetics have been studied extensively6. Unless for very simple examples, in case of secondary amides, the amide bond is predominantly present in the trans- conformation (e.g the ratio of cis / trans is already 0/100 for the simple combination of a methyl and ethyl group as R and R`). The isomerization rate appears to be very slow, even at a higher temperature, as a result of the high activation energy for rotation about the C-N bond. On the contrary, for tertiary amides having similar R` and R`` groups, approximate equimolar mixtures of cis and trans- forms are present7. Moreover, the activation energy required to carry out a rotation around the C-N bond is lower than in secondary amides because of the higher energy level of the tertiary amide rotamers compared to the energetically more stable trans-secondary amide rotamer due to sterical effects of R/R`/R``, while the corresponding transition states have a comparable energy8. This issue of amide conformational restriction has been reported to limit cyclization feasibility in medium-sized rings. As an example, the group of Van Maarseveen9, found that the seven-membered IV.27, (figure IV.7) can be formed from tertiary amide IV.28 as opposed to IV.26 which is attributed to the presence of the trans- conformation of secondary amide in IV.25 on the peptide bond of the precursor.

Part 2: Results and discussion

R O O O N O O H2N H N N OH EDC/HOBt EDC/HOBt 2 N OH H N Bn DMF/CH Cl H DMF/CH2Cl2 2 2 O

IV.25 IV.26 (R=H) IV.28 IV.27 (R=Bn)

Figure IV.7 Example of the influence of a secondary or tertiary amide bond on the formation of homodiketopiperazines IV.26 and IV.27.

Applied to our strategy (figure IV.8), to achieve a successful ring closure, in IV.29 an internal tertiary amide needs to be present; allowing the necessary cis-conformation, since the alternative trans-conformation causes a particularly unfavorable dimensional arrangement of the corresponding reaction partners.

H2N O R3 cis / trans - isomerization O R3 O NH2 O N O O N only for R4 = H R2 R4 R2 R4 R5 cis - IV.29 trans - IV.29

no cyclization only for R4 = H O H N R2 R5 R N 3

O R4

IV.2

Figure IV.8 Cis-/trans- isomerization in tertiary amides IV.29 and their impact on cyclization.

C.2 Effect of α-H atoms in position C-3:

Based on the above, a solid phase synthesis was applied in our laboratory in order to obtain N5- benzylated model compound IV.34 through the resin-bound precursor IV.30. Cyclization using acids, lewis acids, bases or nucleophiles were tested and failed to afford the cyclized IV.34. A

Part 2: Results and discussion minute amount (5%) of the intended benzodiazocinedion could be observed when treating resin- bound precursor IV.30 with t-BuOK (strong base), in addition to a major elimination product (95%) IV.31, while the use of NaOiPr or NaOMe could not alleviate the elimination problem and additionally gave transesterification products. The observation of a small amount of cyclized product seems to result from the deprotonation of the anthranilamine moiety, leading to a more nucleophilic species. Unfortunately the presence of acidic α-hydrogen prevents the use of these conditions for successful library construction.

O O NH2 88% DIC or HO N DCC-PS 82% TFA Bn IV.33 X H O O O NH2 N OH O N H Bn N O O Bn IV.16 IV.30 IV.34 Wang resin KOtBu

NH2 O H + N O Bn O

IV.31 IV.32

Figure IV.9 Formation of by-product IV.31: elimination under strongly basic conditions.

Therefore a solution-phase cyclization was investigated, in which TFA-cleavage of IV.30 was followed by cyclization of IV.33 with the aid of DIC or polystyrene-bound DCC to afford VI.34. Based on this methodology, a simple small library was achieved5 (SPS yields: 15-91% and cyclization yields: 27-90%). The main problem of this cyclization seems to be the formation of N- acyl urea side products arising from O→N acyl urea rearrangement after carbodiimide activation.

Part 2: Results and discussion

O O O NH2 H SPS N HO N DCC OH X R N X O R

Wang resin 15-91% 27-90% IV.16 IV.35 IV.36

Scheme IV.1 Library prepared by solution-phase cyclization using DCC resin.

The successful use of KOtBu-mediated cyclization was demonstrated for resin-bound precursors IV.37 (which do not contain acidic α-protons) leading to a small library of 3,3-dimethyl-1,5- benzodiazocine-2,6-diones IV.38 (yields: 4-58% with crude purity 60-98%)5.

O O O NH H SPS 2 N OH O N KOtBu in THF X R N X O R Wang resin yields: 4-58% IV.16 IV.37 IV.38

Scheme IV.2 Cyclization of resin bound precursor in KOtBu in THF to form IV.38.

IV.3 SPECIFIC AIMS OF THIS WORK

As already mentioned, this project forms the continuation of the above previous work at our laboratory. The specific aims of this PhD can be summarized as follows (figure IV.10):

GOAL 1: In a first part, the synthesis of model compound IV.34 will be repeated, and the synthesis will be optimized where possible. Especially, the cyclization step will be further investigated. Until now, only carbodiimide-based activation was explored in this reaction, leading to less than optimal yields because of unwanted O→N acyl migration side reactions.

Part 2: Results and discussion

GOAL 2: From the resulting optimal conditions, further libraries of structurally diverse 1,5- benzodiazocine-2,6-diones will be constructed using the solution-based cyclization approach; diversity at the 3- and 4-positions will be examined. To this end, anthranilic acid building blocks IV.42 will be used on one hand; on the other hand skeletal diversity at 3- and 4- position will arise from β2- and β3-amino acid building blocks, respectively.

GOAL 3: Lastly, we wish to extend the cyclization/release methodology using strong base-induced cyclization to more complexly-substituted derivatives IV.41 from appropriate β2,2- amino acids

GOAL 4: As the required β-amino acids are generally not commercially available, an in-house synthesis will be necessary.

Part 2: Results and discussion

GOAL 1 O O O NH2 H N HO N Optimization OH Bn N O Bn Wang resin model compound IV.16 IV.33 IV.34 GOAL 2

O O H R2 N R SPS + cyclization NH2 HO 2 + R R4 4 HN COOH N in solution R3 O R 3 IV.42 IV.43 IV.39

O H O N NH HO SPS + cyclization 2 R4 R4 + R2 HN R2 N in solution COOH R3 O R3 IV.40 IV.42 IV.44

GOAL 3

O H O R N R2 2 KOtBu-induced NH2 HO R R3 R5 3 R4 + cyclization/release HN N COOH methodology R4 O R4 IV.41 IV.42 IV.45

GOAL 4

Figure IV.10 Goals of this PhD-research project. The chosen target compounds are shown and their respective basic building blocks.

In the next chapters, a solid phase synthesis route of model compound IV.34 will be discussed intensively, followed by building blocks synthesis and solid phase synthetic route for each representative target compound IV.39, IV.40 and IV.41.

Part 2: Results and discussion

References

1 Blaney, P.; Grigg, R.; Sridharan, V. Chem. Rev. 2002, 102(7), 2607-2624.

2 a) Wilson, L. J.; Li, M.; Portlock, D. E. Tetrahedron Lett. 1998, 39(29), 5135-5138. b) Blass, B. E.; Srivastava, A.; Coburn, K. R.; Faulkner, A. L.; Janusz, J. J.; Ridgeway, J. M.; Seibel, W. L. Tetrahedron Letters. 2004, 45(6), 1275- 1277. c) Li, M.; Wilson, L. J. Tetrahedron Letters 2001, 42(8), 1455-1458.

3 a) Pathak, R.; Roy, A. K.; Batra, K. S. Tetrahedron Lett. 2005, 46(32), 5289-5292. b) Guo, T.; Adang, A. E. P.; Dong, G.; Fitzpatrick, D.; Geng, P.; Ho, K. K.; Jibilian, C. H.; Kultgen, S. G.; Liu, R.; McDonald, E.; Saionz, K. W.; Valenzano, K. J.; van Straten, N. C. R.; Xie, D.; Webb, M. L. Bioorg. Med. Chem. Lett. 2004, 14(7), 1717-1720. c) Mieczkowski, A.; Kozminski, W.; Jurczak, J. Synthesis 2010, 2, 221-232.

4 a) Smith, R. A.; Bobko, M. A.; Lee, W. Bioorg. Med. Chem. Lett. 1998, 8(17), 2369-2374. b) Mayer, J. P.; Zhang, J.; Bjergarde, K.; Lenz, D. M.; Gaudino, J. J. Tetrahedron Lett. 1996, 37(45), 8081-8084.

5 Caroen J., Ontwikkeling van een vastefasesynthesestrategie voor 1,2,3,4,5,6-hexahydro-1,5- benzodiazocine-2,6-dionen voor toepassing in combinatorische bibliotheken, (Development of a solid- phase synthesis strategy for 1,2,3,4,5,6-hexahydro-1,5-benzodiazocine-2,6-diones for application in combinatorial libraries) 2012. PhD dissertation, UGent.

6 LaPlanche, L. A.; Rogers, M. T. J. Am. Chem. Soc. 1964, 86(3), 337-341.

7 LaPlanche, L. A.; Rogers, M. T. J. Am. Chem. Soc. 1963, 85(23), 3728-3730.

8 Gilon, C.; Dechantsreiter, M.A.; Burkhart, F.; Friedler, A.; Kessler, H. “Synthesis of N-Alkylated Peptides” Methods of Organic Chemistry (Houben-Weyl), volume E22c (Synthesis of Peptides and Peptidomimetics), Goodman, M. (ed.), Georg Thieme Verlag, New York, 2003, pp 215-271.

9 Bieräugel, H.; Schoemaker, H. E.; Hiemstra, H.; Van Maarseveen, J. H. Org. Biomol. Chem. 2003, 1, 1830-1832.

Part 2: Results and discussion

V SOLID PHASE SYNTHESIS OF A MODEL 1,5-BENZODIAZOCINE-2,6-DIONE

As this work is the continuation of previous work performed at our laboratory1, the earlier developed solid phase methodology forms the base of the current project. In this chapter the synthetic approach is demonstrated for a model compound 5-benzyl-3,4-dihydro-1,5- benzodiazocine-2(1H),6(5H)-dione and further optimization efforts are explored where necessary.

V.1 SELECTION OF THE SOLID SUPPORT

Wang resin II.4 as a hydroxyl substituted polystyrene resin is chosen for current synthesis as it is available at low cost in a variety of loadings. It allows to cleave off the intermediates after each reaction step in concentrated trifluoroacetic acid solution (TFA). For simplification in this thesis the annotation V.1 is further used.

O OH PS II.4 V.1

Figure V.1: Structure of Wang resin II.4 and a simplified representation V.1 (PS=polystyrene).

V.2 COUPLING OF FMOC-β-AMINO ACID ON WANG RESIN AND FMOC REMOVAL

Coupling of Fmoc-β-alanine on Wang resin was done using classical (Steglich) conditions; preactivation of the carboxylic acid with diisopropylcarbodiimide (DIC) is followed by ester formation in the presence of a catalytic amount of DMAP2, (scheme V.1, step 1). No reaction is observed with the use of other "classical" (amide bond forming) coupling reagents (DIC / HOBt, HBTU, PyBrOP)1. Any unreacted remaining alcohol moities are protected via "capping" with an acetyl group by treatment with an excess of acetic anhydride, (scheme V.1, step 2). At this stage, the loading was determined via UV Fmoc-quantitation (see chapter II). The yield of the final product will be calculated from this value. Typical loading is around 0.6 mmolg-1.

Part 2: Results and discussion

O 1) a) 2 eq Fmoc--alanine, 2 eq DIC, 20 min, CH Cl , 0°C. OH 2 2 O NHFmoc b) 0.2 eq DMAP, CH Cl , r.t, 24h. 2 2 OH

V.1 V.2

2) Ac2O/DIPEA/CH2Cl2 (1/1/3), r.t, 2 x 1h.

O NHFmoc

OAc V.3

Scheme V.1: Coupling of Fmoc-β-alanine to Wang resin.

Fmoc removal with 4-methylpiperidine in DMF delivered the resin V.4, (scheme V.2) with a free primary amine functional group, which allows further attachment with the next building blocks.

O O 20% 4-methylpiperidine in DMF, O NHFmoc 2 x 20 min, rt O NH2

V.3 V.4

Scheme V.2: Fmoc removal using a solution of 20% 4-methylpiperidine in DMF.

V.3 MITSUNOBU-FUKUYAMA ALKYLATION SEQUENCE

In this proposed synthesis, diversity is introduced to the N-5 position via alkylation. In the literature, the difficulty of mono-alkylation of primary amines is a long recognized problem which was also observed by us in solid-phase syntheses (e.g. N-overalkylation using reductive alkylation with aldehydes). To overcome this issue of overalkylation, a stepwise so-called Mitsunobu- Fukuyama alkylation has been developed. The reaction sequence consists of three main steps; 1) amine protection with nosyl chloride, 2) alkylation with alcohol and 3) thiol-mediated deprotection of the alkylated product. The utility of this methodology is a result of the presence of an acidic

Part 2: Results and discussion sulfonamide proton (prerequisite for Mitsunobu reaction) and secondly, the simple removal of the carefully selected sulfonamide by a nucleophilic aromatic substitution reaction 3,4.

V.3.1 Step 1: Protection with nosyl group:

Treating the resin-bound amino acid V.4 with excess 2-nitrobenzenesulfonyl chloride (conc: 0.2

M in CH2Cl2) in the presence of collidine provides cleanly the resin-bound protected amino acid V.5, as evidenced by negative (=colorless) TNBS test for amines and by LCMS analysis of the crude product obtained by cleaving an aliquot of resin with TFA.

NO2 , 10 eq O 5eq O O NO2 SO Cl N O 2 S O NH O N 2 H CH2Cl2, r.t, 2x1h. V.4 V.5

Scheme V.3 Protection of the resin-bound amino acid with o-nosyl group.

V.3.2 Step 2: Mitsunobu Reaction

At this stage, the actual Mitsunobu alkylation of the sulfonamide can take place using benzyl alcohol through a bimolecular nucleophilic substitution (SN2) reaction after activation with triphenyl phosphine and diisopropylazodicarboxylate5, scheme V.4.

O NO2 O NO O O 10 eq BnOH, 5 eq Ph P, 5 eq DIAD, O O 2 S 3 S O N DCE, rt, 3 x 2 h O N H Bn

V.5 V.6

Scheme V.4 Mitsunobu alkylation of V.5 with benzyl alcohol.

Complete alkylation is achieved after triple treatment for 2 h, as monitored by LCMS analysis of TFA-cleaved intermediate.

Part 2: Results and discussion

VI.3.3 Step 3: Removal of nosyl group

Removal of the 2-nitrobenzenesulfonyl is accomplished by reaction with 2-mercaptoethanol in presence of the base DBU. The reaction proceeds via a nucleophilic aromatic substitution in which the electron deficient aromatic ring undergoes a nucleophilic attack of the thiolate anion and the thus formed Meisenheimer complex (V.8, scheme V.6) after elimination affords the desired mono- alkylated secondary amine V.7.

O O NO2 O O OH S 5 eq HS , 2.5 eq DBU, DMF, O N O NH Bn r.t, 2 x 30 min Bn

V.6 V.7

Scheme V.5 Removal of the nosyl group by 2-mercaptoethanol and DBU.

O O NO2 O O O O O S S NO O N S 2 O N O NH + SO2 Bn Bn Bn

V.6 Meisenheimer S OH complex V.8 V.7

NO2 S + HO

V.9 Scheme V.6 Mechanism for the deprotection of sulfonamide V.6.

V.4 COUPLING OF FMOC-ANTHRANILIC ACID DERIVATIVES

In the literature, there are several problems observed around the coupling of secondary amines to carboxylic acids6. Even though these secondary amines may be expected to display a higher nucleophilicity than their primary counterparts, it appears that the steric hindrance has an important influence during amide coupling reactions. Many conditions using a range of coupling reagents

Part 2: Results and discussion and additives have been previously tested in our laboratory for this transformation. It turned out that only activation of the carboxylic acid as anhydride could afford the desired resin-bound amide in good yields, in agreement with literature reports. Thus, the reaction of 10 eq N-Fmoc-anthranilic acid with 5 eq diisopropylcarbodiimide (DIC) yields the symmetrical anhydride V.11, which is then allowed to react with resin-bound secondary amine V.7. A full conversion was observed after 24 hours treatment (concentration anhydride: 0.2 M).

O O NHFmoc O O N OH

NHFmoc

V.10 V.12

Preactivation Step Coupling Step a) b) O O FmocHN O O NHFmoc BnHN V.7 (1 eq) O

V.11

a) 10 eq V.10 , 5 eq DIC, CH2Cl2/DMF 9/1, 20 min, r.t, 0 C. b) r.t, 24h.

Scheme V.7 Coupling of a secondary amine with an anthranilic acid in two steps; preactivation to symmetrical acid anhydride then coupling to primary resin-bound amine.

The use of halogenated solvents is necessary for successful reaction, which has been hypothesized to occur via intermolecular hydrogen bonding during the nucleophilic addition step (figure V.2)7. The use of a polar solvent would increase competitive solvent hydrogen bonding, thereby slowing down the reaction considerably. Because of the poor solubility of Fmoc protected anthranilic acid derivatives and their symmetrical anhydride in halogenated solvents, the reaction is typically performed in CH2Cl2/DMF 9/1.

Part 2: Results and discussion

O O

"R O R" O O R' V.14 R' N "R N + "R OH R H CH2Cl2 R

V.13 V.15 V.16

O O

"R "R R' O R' O H R" H R" N O N O

R R

V.17 V.18

Figure V.2: Hypothesized reaction mechanism between a secondary amine and a symmetrical acid anhydride in halogenated solvent.

V.5 FMOC REMOVAL AND CLEAVAGE FROM RESIN

Before we could perform the cyclization reaction, the amine had to be deprotected (scheme V.8). This Fmoc removal was achieved by shaking the resin V.12 in a solution of 20% 4- methylpiperidine in DMF for 20 min. The reaction is repeated once to afford V.19.

Part 2: Results and discussion

O O NHFmoc O O NH2

O N O N 20 % 4-methylpiperidine in DMF, 2x 20 min

V.12 V.19

TFA/H2O 95/5, 1 h

O O NH2 HO N

(88% from V.3)

V.20

Scheme V.8 Fmoc removal using a solution of 20% 4-methylpiperidine in DMF and cleavage from

Wang resin in a mixture of TFA/H2O 95/5.

Cleavage from the resin is done by treatment of the resin with a mixture of TFA/H2O 95/5 for 1 h to obtain the ring closing precursor V.20 in solution (scheme V.8). Purification of the crude material (crude purity: 95%, 214 nm) by column chromatography gave the pure dipeptide with an isolated overall yield of 88% (calculated from V.3).

V.6 CYCLIZATION IN SOLUTION

An overview of attempted cyclization conditions is displayed in scheme V.9 and table V.1. Reactions were typically performed on small scale and monitored by LCMS. Promising conditions were then repeated on larger scale, for which isolated yields are reported.

Part 2: Results and discussion

O O O NH H 2 N HO N a) coupling reagents (V.28-V.36) and solvent (see table V.1), shaking 1 hour, r.t. N O

V.20 IV.34

Scheme V.9 Cyclization step using different conditions.

Table V.1: Results for cyclization reaction with coupling reagents V.28-V.36.

Exp reagent (a) Eq Solvent results(b) Purity(c)

18 ClCOOEt 1.1 Dioxane Product

Et3N 1.2 (48% isolated yield) 2 ClCOOMe 2 Dioxane Product + starting material 85% DIPEA 4 84%+1% 3 ClCOOMe 2 DCM/DMF Product 94% DIPEA 4 95/5 94% (75% isolated yield) 41 DCC resin 2 DCM Product + cyclic dimer 89% 1.9 mmol/g 89%+2% 5 DIC 2 DCM/DMF Product + N-acylurea 35% DIPEA 4 95/5 35% +58%

6 DIC 2 DCM/DMF Starting material + product + 54% HOBt 2 95/5 N-acylurea 1% +53% +13%

7 DIPEA 2 DMF Product + HOBT 74% HBTU 2 74% + 15%

8 Pivaloyl chloride 2 DCM/DMF Starting material + product + 35% DIPEA 4 95/5 N-Pivaloyl side product 1% + 34% + 20%

Part 2: Results and discussion

9 BOP 2 DMF Starting material + product + 70% DIPEA 4 mixture 2% + 68%

10 PyBrop 2 DMF Starting material + product + 57% DIPEA 4 mixture 1% + 56% 11 TFFH 2 DMF Starting material + product + 50% DIPEA 4 O-acylurea (mixture) 4% + 46% (a) The concentration of the coupling reagent is 0.01M (b) Relative ratio determined from the peak areas in the HPLC chromatogram at 214 nm of the corresponding compounds unless specified otherwise; (c) Total HPLC purity of starting material and end product in the crude mixture (214 nm).

The cyclization of V.20 to IV.34 was literally described by Gatta8 (scheme V.10) using ethyl chloroformate to activate the carboxylic acid, giving the desired product with a reported yield of 63%. However, in our hands we achieved lower yields (48%). Upon using DIPEA as an alternative base (table V.1, experiment 2) and changing solvent to DCM/DMF 95/5 (table V.1, experiment 3) LCMS analysis of crude reaction mixture showed increasing amounts of product (85%/94%; relative purity determined via LCMS at 214 nm). Repeating the experiment (experiment 3) led to an isolated yield of 75%.

H O O O NH2 1.1 eq ethyl chloroformate, O O O NH2 N 1.2 eq Et3N, 1,4-dioxane, HO N r.t, 1 h O O N N 48% (63% according to Gatta) O

V.20 V.21 IV.34 Scheme V.10 Ring closure of V.20 according to reported Gatta8 conditions.

A more successful ring closure was achieved at our laboratory1 through the resin-bound dicyclohexylcarbodiimide (DCC) coupling reagent (scheme V.11), giving a clean reaction mixture, leading to the purified compound in 75% yield. According to LCMS-analysis, the crude

Part 2: Results and discussion product was contaminated with a low amount of cyclic dimer V.23 (figure V.3). The loss of the remaining (25%) material was attributed to the occurrence of an O→N acyl migration of the DCC- resin-activated carboxylic acid. This O→N acyl migration was confirmed by treatment of V.20 with DIC, giving the N-acylurea derivative V.27 (scheme V.12, table V.1, experiment 5).

DAD1 A, Sig=214,20 Ref=off (H:\TEMP\069-4401.D) mAU

1750 4.724 O O NH 1500 O HN N

1250 N 1000 N O O HN

750 O IV.34 500 desired product V.23 cyclic dimer (2%) 250

6.149

0.964

2.681

0.917 1.502

1.630

1.229

2.727 0 1.085

3.525

3.632

3.969

4.202

4.283

5.060

5.332

6.311

8.906

6.870

9.500

0 2 4 6 8 min Figure V.3: LC-MS spectrum reported by a DAD detector at 214 nm after the cyclization reaction.

H O N O O NH2 O N CH2Cl2 N C N + HO N NH H2N 2h, r.t. O activation 2 eq V.36 V.20 V.22

ring closure

O O NH O O N HN HN NH

16 + + N N O HN O O

V.23 IV.34 V.24 cyclic dimer 75% filtered

Scheme V.11: Cyclization of V.20 using resin-bound DCC.

Part 2: Results and discussion

Side reaction: using PS-DCC

O O NH2 O O O NH2 N PS-DCC O N O N acyl migration V.20 N N N H NH

V.22 V.25 resin-bound by-product using DIC:

O O NH2 O O O NH2 N DIC O N O N acyl migration N N N V.20 H H2N

V.26 V.27 Scheme V.12 By-products formed using DCC resin or DIC as a coupling agent due to O→N acyl migration

In an attempt to optimize the yields of compound IV.34, further conditions have been tested for cyclization, using a variety of coupling reagents and additives (structures, see figure V.4).

Where treatment of V.20 with DIC affords 35% (relative purity determined via LCMS at 214 nm) of the 1,5-benzodiazocine in addition to 58% of N-acyl urea V.26 (experiment 5), the amount of the latter could be decreased by adding the additive HOBT (experiment 6). Using HBTU as the coupling agent, 74% of product can be obtained. However, performing the reaction in DMF is not desirable due to difficulty of removing it from reaction (experiment 7). Using pivaloyl chloride (experiment 8), with 4 eq of DIPEA, led to a lower percentage of cyclized product besides 20% of the product resulting from N-pivaloylation.

Part 2: Results and discussion

H N N O N N N C N CH N N 3 Cl O N OH CH3 PF 6 N CH3 H3C

DIC HOBT HBTU Pivaloyl Chloride V.28 V.29 V.30 V.31

N N PF6 N N N N Br P N O P F PF6 N N N N PF6

BOP PyBrOP TFFH V.32 V.33 V.34

N C N N O

O O

IIDQ-PS DCC-PS V.35 V.36

Figure V.4: Structures of the used coupling reagents and additives.

The use of BOP reagent in presence of DIPEA affords the product in moderate purity. PyBrOP in presence of DIPEA affords lower percentage of product in addition to mixture of compounds (56% purity). The use of TFFH to activate the carboxylic acid as acid fluoride affords lower percentage of product and traces of the open form in addition to unexplained mixture of compounds (purity 50%).

Based on these experiments, both the use of carbodiimide or ClCOOMe-based activation of V.20 seems to deliver the best cyclization results. Although also effective, the HBTU method is not

Part 2: Results and discussion preferred considering the use of DMF and the equimolar formation of HOBt, which has to be removed using chromatography.

Although the isolated yield of both DCC resin and ClCOOMe is 75%, the work up of DCC resin is easier than ClCOOMe, as the higher temperature required to remove DMF used in the latter could lead to decomposition of cyclized product. The formation of 2 % of 16-membered ring next to 8-membered ring occurs during the cyclization with DCC resin, while no observation of such 16-membered ring using ClCOOMe.

Syntheses based on solid carrier-bound reagents are gaining more popularity in comparison with pure solid-phase synthesis strategy, especially since the benefits of solution phase chemistry remain available (easy reaction monitoring, intermediate purification; if necessary) surmounted by a simple finishing procedure (in the ideal case: filtering off the solid phase reagents and / or scavengers, extraction of the product and evaporation of the volatiles).

The overall yield of the model 1,5-benzodiazocine-2,6-dione IV.34 calculated from resin-bound N-Fmoc-β-Alanine V.3 is 67% (8 steps). This yield is encouraging to undertake the synthesis of libraries of 3- and 4-substituted analogous. Our effort in this respect are discussed in the following chapters.

Part 2: Results and discussion

References

1 Caroen J., Ontwikkeling van een vastefasesynthesestrategie voor 1,2,3,4,5,6-hexahydro-1,5- benzodiazocine-2,6-dionen voor toepassing in combinatorische bibliotheken, (Development of a solid- phase synthesis strategy for 1,2,3,4,5,6-hexahydro-1,5-benzodiazocine-2,6-diones for application in combinatorial libraries) 2012. PhD dissertation, UGent. 2 a) Mergler, M.; Tanner, R.; Gostelli, J.; Grogg, P. Tetrahedron Lett. 1988, 29, 4005-4008; b) Pedroso, E.; Grandas, A.; Saralegui, M. A.; Giralt, E.; Granier, C.; Shots of Reed J. "Convergent solid phase peptide synthesis. I. Synthesis of protected segments.

3 Lin, X.; Dorr, H.; Nuss, J. M. Tetrahedron Lett. 2000, 41, 3309-3313.

4 Kan, T.; Fukuyama, T. Chem. Commun. 2004, 4, 353-359.

5 Swamy, K. C. K.; Kumar, N. N. B.; Balaraman, E.; Kumar, K. V. P. P. Chem. Rev. 2009, 109(6), 2551- 2651.

6 Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97 (6), 2243.

7 Fu, Y.; Hammarström, L. G.; Miller, T. J.; Fronczek, F. R.; McLaughlin, M. L.; Hammer, R. P. J. Org. Chem. 2001, 66 (21), 7118-7124

8 Gatta, F.; LandiVittory, R. Farmaco, Ed. Sci. 1970, 25, 991-998

Part 2: Results and discussion

VI SYNTHESIS OF 3-SUBSTITUTED-1,5-BENZODIAZOCINE-2,6-DIONES

VI.1 RETROSYNTHESIS

In this chapter we would like to discuss the synthesis of 3-substituted-1,5-benzodiazocine-2,6- dione derivatives. After a retrosynthetic analysis, the synthesis of necessary building blocks is described after which the application of these to the construction of a library is treated.

As been described in chapter IV, ring-closure is planned through lactam formation from precursor VI.2, in which a second amide bond can be disconnected, affording two general building blocks: the anthranilic acids VI.4 and the resin-bound β-amino acids VI.3. A series of (unprotected) anthranilic acids is commercially available while the resin-bound β-amino acids VI.3 can be synthesized from β-amino acid building blocks VI.5 and alcohols R2OH.

NHFmoc HOOC

R3 anthranilic acids

VI.4 O H + N lactam R1 O O NH2 amide bond O formation formation R3 O N O NH N R R R R O R 1 2 1 2 2 R3 VI.1 VI.2 VI.3 -amino acid coupling and N- alkylation (R2OH)

OH + HO NHFmoc

R1 -amino acid VI.6 VI.5

Figure VI.1: Retrosynthesis for target benzodiazocinedione VI.1.

Part 2: Results and discussion

VI.2 BUILDING BLOCK SYNTHESES

VI.2.1 Synthesis of N-Fmoc-anthranilic acid building blocks

VI.2.1.1 Direct protection

For the intended solid-phase synthesis route, the use of N-protected anthranilic acid building blocks is necessary. We opted for the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group, as it is well established in solid-phase synthesis literature.

Although N-Fmoc-anthranilic acid is commercially available, it is too expensive when needing greater amounts. The protection step, however, is very simple: by using equivalent amounts of anthranilic acid and 9-fluorenylmethyl-N-succinimidyl carbonate (Fmoc-OSu) in an aqueous basic environment, large scale synthesis is possible (scheme VI.1). These reaction conditions were successfully applied for the synthesis of aryl-substituted analogues (VI.7-VI.11) which are useful building blocks for library synthesis; yields are provided in table VI.1.

3 4 2 NHFmoc NH2 i) 1 eq NaOH, H2O, r.t, 5 min R R 1 ii) 1 eq NaHCO3, 1 eq Fmoc-OSu, 5 COOH 6 COOH THF/H2O 2/1, r.t, o.n

2-aminobenzoic acid Fmoc-2-aminobenzoic acid

NHFmoc NHFmoc NHFmoc

COOH Br COOH MeO COOH

VI.7 VI.8 VI.9 NHFmoc NHFmoc

COOH Me COOH F VI.10 VI.11

Scheme VI.1 Synthesis of N-Fmoc-2-aminobenzoic acid derivatives.

Part 2: Results and discussion

Table VI.1: Yields of the prepared anthranilic acid derivatives.

Entry Compound R Yield %a

1 VI.7 H 85

2 VI.8 5-Br 83

3 VI.9 5-OMe 63

4 VI.10 5-Me 60

5 VI.11 6-F 57 a Yields are calculated after recrystallization .

To increase diversity, we also envisaged the use of 5-phenyl anthranilic acid, which would contain a biphenyl moiety, a well-known privileged substructure. However, since the compound is not commercially available, we tried different synthetic routes to prepare it, which is discussed in the following paragraphs.

VI.2.1.2 Attempts to the synthesis of 5-phenyl anthranilic acid

VI.2.1.2.1 Via Baeyer-Villiger oxidation of 5-phenyl isatine:

A- Starting from 4-amino biphenyl:

Inspired by the known conversion of anilines to their isatin derivatives and the subsequent oxidation/hydrolysis to the anthranilic acid derivatives, we initially started from 4-amino biphenyl VI.12. Although the reaction with chloral hydrate in presence of excess hydroxylamine hydrochloride yielded the oxime VI.13 in moderate yield1, treatment of VI.13 in strong acidic medium, as is commonly used for isatin derivatives synthesis, surprisingly did not afford 5-phenyl isatine (scheme VI.21). Instead, starting material remained as major product in addition to a mixture of compounds.

Part 2: Results and discussion

H H NH2 N O Chloral hydrate, NH2OH.HCl H SO N 2 4 O 60-80 C Ph Na2SO4, Conc. HCl, H2O, 80C Ph N H Ph 76% O OH VI.12 VI.13 VI.14

NH NHFmoc 1) Baeyer- 2 Villiger Oxidation 2) hydrolysis Ph COOH Ph COOH VI.15 VI.16

Scheme VI.2 Initially tried synthetic route through the formation of anilide VI.13.

B- Starting from 5-iodoisatine:

According to a one pot procedure by Rault2, Suzuki cross-coupling reaction of 5-iodoisatine VI.17 with phenylboronic acid under palladium catalysis, followed by oxidative cleavage with H2O2, would afford 5-phenyl anthranilic acid VI.15 in 62%. However, application of these conditions only allowed us to isolate the expected product in 15% yield (scheme VI.3).

H H Baeyer-Villiger NH2 N N Pd(PPh3)4, PhB(OH)2, NaHCO3, DME, Oxidation O O H2O, 0.2 M, reflux, Ar, o.n. Ph COOH I Ph NaOH/H2O 15 % O O H2O2 (30%) reflux 30min. VI.17 VI.18 VI.15 (One pot reaction)

Scheme VI.3 One pot reaction sequence to form 5-phenyl anthranilic acid2 VI.2.

We decided to split the reaction sequence, and focus on the Suzuki-coupling. Chen3 used similar conditions to Rault, albeit under microwave irradiation in order to a obtain library of 4-aryl isatins. When we apply the same conditions on 5-iodoisatin VI.17, we obtained a product which we cannot assign, (table VI.2, experiment 1). Dawei4 succeeded in preparing a series of 7-aryl substituted isatins from 7-iodoisatins using KF or NaHCO3 as base, when using substituted boronic acids. Application of these conditions for 5-iodoisatin VI.17 (experiment 2, 3) did not provide 5-phenyl isatin, as only starting material was recovered.

Part 2: Results and discussion

7 H H 1 N 6 N 2 see table IV.2 O O 5 3 I 4 Ph O O VI.17 VI.18

Scheme VI.4 Cross-coupling alkylation attempts to form 5-phenyl isatin.

Table VI.2 Conditions tested in cross-coupling of 5-iodoisatin and phenyl boronic acid.

Exp Reagents Solvent T Δt Results

1 0.1 eq Pd(PPh3)4 (5mol%), DME/H2O Mw 30 min Not assigned

1.2 eq PhB(OH)2, 2 eq 0.06 M 130 °C product

NaHCO3

2 0.06 eq Pd(OAc)2 (3mol%), MeOH Reflux o.n Starting

1.5 eq PhB(OH)2, 3 eq KF 0.06 M 60 °C material

3 0.07 eq Pd(PPh3)4, 1.2 eq DME/H2O Reflux o.n Starting

PhB(OH)2 (5mol%), 2 eq 0.06 M 100 °C material

NaHCO3

V.2.1.2.2 Ortho functionalization starting from 4-amino biphenyl

An alternative strategy to synthesize anthranilic acids involves the ortho-carboxylation of amide- or carbamate- protected anilines using an ortho-lithiation procedure5. Boc-protection of 4- aminobiphenyl VI.12 proceeds easily with high yield to give VI.19, which should be a good substrate for ortho-lithiation and conversion to anthranilic acid derivative VI.20.

Part 2: Results and discussion

NH2 NHBoc NHBoc 1 eq Boc2O, 1eq DIPEA, see table VI.3

Ph CH2Cl2 0.1 M, 0 C-r.t, o.n Ph Ph COOH 90% via VI.1 VI.19 VI.20

N OtBu

O Ph

VI.21

Scheme VI.5 Attempted synthesis of N-Boc-5-phenylanthranilic acid via ortho-lithiation. Different tested conditions are mentioned in table VI.3.

After formation of the (yellow-orange) dianion VI.21 using tBuLi, in experiment 1, the reaction mixture was added to a slurry of CO2, resulting in no reaction. Alternatively, a solution of CO2 in THF was added during 2 hours over the generated dianion resulting in a very small amount of unexplained products, (experiment 2)6. Stirring the reaction for an additional 5 hours and increasing the temp to room temperature results in no reaction, (experiment 3)7. Increasing the concentration of reaction, on the other hand provides a small amount of the product (10%), next to 15% of Boc-deprotected 5-phenyl anthranilic acid VI.15 in addition to starting material VI.19 as major.

Table VI.3 Tested reaction conditions for ortho-lithiation and reaction with CO2.

Exp Reagents Solvent T Δt Results 1a 2.4 eq (1.7M) Tert- THF -20 °C 2 hours Starting material

Butyllithium, CO2 0.5M 2b 2 eq (1.7M) Tert- THF r.t 2 hours bubbling 95%start+5%unknown

Butyllithium, CO2 0.4M product

3c 3 eq (1.7M) Tert- THF r.t 2hours bubbling Starting material

Butyllithium, CO2 0.1M + 5hours stirring 4c 2 eq (1.7M) Tert- THF r.t 40 min bubbling 70% starting material +10%

Butyllithium, CO2 0.4M product + 15% VI.15 a: reaction mixture was added to slurry of CO2, b: solution of CO2 in THF was added to reaction mixture via cannula, c: CO2 was passed over CaCl2 tube to the reaction mixture.

Part 2: Results and discussion

Because of the encountered problems to provide the desired product in a sufficient yield, we decided at this stage to not perform further optimization experiments and therefore omit it in the library syntheses.

V.2.2 Synthesis of N-Fmoc-3-amino-2-alkylpropionic acid building blocks

The other main building blocks for our solid-phase synthesis approach are N-Fmoc-3- aminoalkylpropionic acids VI.5 as shown in figure VI.1. To test the synthesis towards these building blocks, we decided to access a model building block VI.22 to demonstrate the synthetic steps and apply the optimized conditions to a set of further analogues. In the following sections the search for a general synthetic pathway for β2-(alkyl)-β-amino acid synthesis is discussed.

FmocHN COOH

model compound VI.22

Figure VI.3: 3-(9-Fluorenylmethyloxycarbonyl)amino-2-(4-bromobenzyl)propanoic acid VI.22 as a model compound.

V.2.2.1 Synthesis starting from Boc-β-alanine

Our initial attempted strategy involves the direct introduction of an alkyl group via α-alkylation of protected β-alanine. Previously in our laboratory, in this way β2-methyl-β-amino acid derivative VI.24 could be prepared, using conditions originally reported by the group of Seebach8. The procedure involved the deprotonation of Boc-protected methyl ester of β-alanine VI.23 using LDA, after which treatment with MeI as electrophile yielded the mono-methylated product VI.24 in 58% yield, (unoptimized, additionally 26% of VI.23 could be recovered, scheme VI.6)9. Attempts to generalize this alkylation strategy by applying these conditions to 4-bromobenzyl bromide as electrophile yielded a mixture from which the desired product VI.25 could only be isolated with a very low yield. Reasons for this low yield can be explained by the formation of a complex mixture (from which the product could only be partially purified), probably arising from unwanted N-alkylation and over alkylation.

Part 2: Results and discussion

O O a) 2 eq LDA, THF, -78°C, 20 min. BocHN OMe BocHN OMe b) 4 eq MeI, THF, -78°C, 2h VI.24 VI.23 58% yield (26 % recovered VI.23)

O O a) 2 eq LDA, THF, -78°C, 20 min. BocHN OMe BocHN OMe b) 4 eq 4-bromobenzylbromide, THF, -78°C, 2h Br

VI.23 VI.25 6%

Scheme VI.6: Direct alkylation of protected β-alanine VI.23 with LDA and 4-bromobenzyl bromide.

VI.2.2.2 Synthesis starting from methyl cyanoacetate

To avoid undesired N-alkylation, the alternative alkylation of methyl cyanoacetate with alkyl halides was proposed (scheme VI.7). However, in the model case of 4-bromobenzyl bromide a major problem of double alkylation occurs, although different conditions were tested (table VI.4).

Br Br Br O + NC a) 4-bromobenzyl bromide VI.29, OMe base (table VI.4) NC COOMe NC COOMe

VI.26 VI.27 VI.28

Scheme VI.7: Attempted direct alkylation of methyl cyanoacetate with bromobenzyl bromide.

Part 2: Results and discussion

Deprotonation using K2CO3 and subsequent treatment with equimolar amounts of bromobenzyl bromide gave no expected product, but only a mixture of bis-benzylated VI.28 and remaining starting material. Use of an excess of methyl cyanoacetate (3 eq, 1 eq 4-bromobenzyl bromide) gave only low amounts of the mono-alkylated product, in which the major components was the bis alkylated species. Changing the solvent (DMF, THF) or inverse addition did not improve on the outcome of the reaction.

Table VI.4. Different conditions used in the direct alkylation with an alkyl halide.

Exp Reagents solvent T Δt Resultsa VI.28/VI.27/VI.26 b 1 1 eq K2CO3 DMF 0°C 1h 63% / 0 / 37% 1 eq 4-Bromobenzyl bromide, 1 eq methyl cyanoacetate 2b 1 eq NaH, DMF 0°C 3h 25% / 19% / excess 1 eq 4-Bromobenzyl bromide, 3 eq methyl cyanoacetate 3b 1 eq NaH, THF 0°C 3h 28% / 14% / excess 1 eq 4-Bromobenzyl bromide, 3 eq methyl cyanoacetate 4c 1 eq NaH, THF 0°C 3h 14% / 7% / 79% 1.5 eq 4-Bromobenzyl bromide, 1 eq methyl cyanoacetate a Based on LCMS by comparing peak areas at 214 nm. b 4-bromobenzyl bromide was added to a solution of methyl cyanoacetate and base. c a solution of methyl cyanoacetate and NaH was stirred for 30 minutes and added dropwise to a solution of 4-bromobenzyl bromide.

Part 2: Results and discussion

VI.2.2.3 Knoevenagel condensation followed by reduction:

To avoid the unwanted overalkylation problems, we searched for an alternative mono-alkylation route, by using a two-step sequence via a Knoevenagel-type condensation. This methodology is based on the initial preparation of a dehydro-cyanoacetate intermediate capable of producing only mono-alkylated product after double bond reduction (scheme VI.8). In this way, structural diversity in the final β2-amino acids would arise from employing a set of different aldehydes, rather than alkyl halides.

1eq 4-Bromobenzaldehyde. 0.1 eq AcOH, 0.04 eq piperidine, dioxane, Pd/C 5% wt, H2 (1bar), 2 h, r.t.

Br Br H O NC i (see table VI.5) + OMe NC COOMe 87% NC COOMe NC COOMe "E" VI.26 VI.30 VI.27 VI.31

i) 1 eq 4-Bromobenzaldehyde. 0.1 eq AcOH, 0.04 eq piperidine, dioxane, o.n., r.t

Scheme VI.8: Reaction scheme to secure mono-alkylated product VI.27 via a Knoevenagel / reduction route.

This methodology has been published as one pot procedure for the reaction between methyl cyanoacetate and unsubstituted benzaldehyde, in which the Knoevenagel reaction was allowed to 10 proceed in the presence of Pd/C under an H2 (1-2 bar) atmosphere . However, after applying this procedure on 4-bromobenzaldehyde at 1 bar H2, unreacted methyl cyanoacetate was still present while unwanted hydro-dehalogenation of the aryl bromide occured (VI.31)11. We therefore opted for a two-pot procedure and as such the application of the reported Knoevenagel conditions

(without Pd/C and H2-atmosphere) afforded the condensed product VI.30 in 87% yield. From LCMS and NMR spectral data it is clear that only one isomer is formed and based on literature evidence12, we assign the “E”-double bond geometry to the isolated product. In order to prevent the hydrogenolysis of the bromide, we tried to explore alternative olefin-reduction methods. Several reaction conditions were tested (table VI.5).

Part 2: Results and discussion

Cl MeO

NC COOMe NC COOMe NC COOMe

VI.32 VI.33 VI.34 85% 88% 64%

NC COOMe NC COOMe

VI.35 VI.36 88% 91%

Figure VI.6: Yields obtained by condensation of methyl cyanoacetate and different substituted aldehydes.

The reducing agent used should be able to reduce the intended double bond but should however, not be too strong in order to avoid the reduction of the nitrile or methyl ester. In experiment 1, the use of NaBH(OAc)3 yielded only a partial conversion while the long reflux conditions in ethanol yielded additionally the ethyl esters of both start and end product. Consequently a stronger reducing agent (NaBH4 in presence of Pd/C) was chosen (experiment 2) according to a published procedure in which the use of a non-polar solvent seems to be important13.

Part 2: Results and discussion

Table VI.5: Tested reaction conditions for the reduction of arylidene VI.30.

Exp Reagents Solvent T Δt Results

1 1 eq VI.30, 5 eq Ethanol reflux 24h product (+ethyl ester)

NaBH(OAc)3 +small fraction VI.30 (+ ethyl ester)

2 1 eq VI.30, 2 eq AcOH, 4 Toluene r.t 18 h product + small fraction

eq NaBH4, 2.5 mol% Pd/C VI.30 + VI.31

3 1 eq VI.30, 2 eq AcOH, 4 Toluene r.t 7h product + VI.30

eq NaBH4, 2.5 mol % Pd/C

4 1 eq VI.30, 1.1 eq Ethanol r.t 45 product (+ ethyl ester)

NaBH3CN, AcOH (0.4 M) min

5 1 eq VI.30, 1.1 eq MeOH/CH2Cl2 r.t 45 product (82% yield) NaBH CN, AcOH (0.4 M) min 3 5/1

Reduction of both the α,β-unsaturated C-C double bonds and the carbonyl group may happen using these reagents, but the authors state that by increasing solvent polarity, the selectivity towards carbonyl reduction increases. This effect is hypothetically explained that if the reaction is carried out in a polar solvent, it dissolves readily NaBH4 and then reacts with the carbonyl via a 1,2- mechanism. The poor solubility in a non-polar solvent would ensure that the NaBH4 would be more likely to react with acetic acid and to produce hydrogen gas that can bind to Pd/C to form a Pd-complex which catalyzes the hydrogenation of double bond. Under these conditions a portion of H-substituted product VI.31 was recovered after 18 hours reaction time, probably as a consequence of the above-mentioned sensitivity of the aromatic bromide. When the reaction time was shortened (7 hours, experiment 3), the unwanted Br-H-substitution could be avoided. However, insufficient conversion of the starting material VI.26 was observed.

14 An article by Hutchins uses NaBH3CN, a reducing agent which is in terms of strength between

NaBH(OAc)3 and NaBH4 for the reduction of double bonds conjugated with two electron-

Part 2: Results and discussion withdrawing groups. Through a slightly modified procedure (AcOH instead of HCl) the desired reduction gives complete conversion. In spite of the short reaction time, a small amount of ethyl ester was formed by the presence of ethanol/AcOH as the solvent. Upon changing the solvent to methanol, solubility problems arise, which could be solved by using a mixture of

(MeOH/CH2Cl2)(5/1), leading to good yield (82%) of VI.27. This two-step Knoevenagel/reduction sequence was successfully applied to a series of derivatives (figure VI.6- VI.7). Although available literature only describes compounds carrying electron withdrawing aryl substituents, we could apply the reduction procedure to electron-rich derivatives (figure VI.7).

Cl OMe

NC COOMe NC COOMe NC COOMe

VI.37 VI.38 VI.39 89% 96% 98%

NC COOMe NC COOMe

VI.40 VI.41 92% 89%

Figure VI.7: Yields obtained by reduction of the corresponding alkenes with NaBH3CN/AcOH in

CH2Cl2/MeOH

Besides the use of aryl aldehydes, we also wanted access to β2-alkyl-β-amino acids. This turned out to be straightforward. Applying the original (one pot knoevenagel condensation/reduction) procedure using isobutyraldehyde (with an increase in the hydrogen pressure to 2 bar), yielded 90% of the target product VI.42, (scheme VI.9).

Part 2: Results and discussion

1 eq isobutyraldehyde, 0.1 eq AcOH, 0.04 eq piperidine, NC COOMe NC COOMe dioxane, 0.1 eq Pd/C 5%wt, 2 bar, 2h VI.26 VI.42 90%

Scheme VI.9: Knoevenagel condensation and in situ reduction to form compound VI.42.

VI.2.2.4 Reduction of nitrile group

The reduction of the nitrile function is classically carried out with H2 under high pressure using

Pd/C as catalyst or with LiAlH4. These two methods are not applicable for us, on one hand because of the expected reduction of the aromatic halides and on the other hand because of the reactivity of the methyl ester15. The tested alternatives are shown in table VI.6.

Br Br Br

(see table VI.6) R + HN NC COOMe COOMe NC COOH

VI.27 VI.43 R=H VI.45 VI.44 R=Boc, 94%

Scheme VI.10: Reduction of the cyano group in VI.27 to the β-aminoacid ester

In experiment 1, the reduction according to the procedure of Baddorey16 using Raney-Nickel led to the formation of a small amount of the desired product VI.43, but the mixture mainly consisted of the starting material VI.27 and corresponding carboxylic acid; which formed due to hydrolysis (coming from the slurry of Raney-Nickel).

Part 2: Results and discussion

Table VI.6: Tested reaction conditions for the reduction of cyano group of VI.27.

Exp Reagents Solvent T Δt Results

1 1 eq VI.27 , Ra-Ni 0.5% NH3 in r.t 24h VI.45 + VI.27 + small

slurry (in H2O) MeOH fraction VI.43

2 1 eq VI.27, NiCl2.6H2O Isopropanol r.t 3h Complex reaction + (saturated), 7.5 eq VI.27

BH3.THF

3 1 eq VI.27, 2 eq Boc2O, MeOH 0°C to r.t 30 min VI.44 + alcohol +

0.1 eq NiCl2.6H2O, 7 eq reduced bromide+ other

NaBH4 products, (49% yield) amonitoring by LCMS (214 nm)

In experiment 2, the reduction is carried out using borane in a saturated NiCl2 solution in isopropanol. Although described in literature for cyanohydrines17, this led to complex reaction mixtures containing unreacted cyanoacetate VI.27. Better results were achieved by application of 18 a procedure reported by Caddick (experiment 3), wherein a mixture of NaBH4/NiCl2 in MeOH is used. By the addition of NaBH4 to NiCl2.6H2O in methanol, hydrogen gas is formed along with black precipitate of Ni2B that is capable of catalyzing the further reaction between NaBH4 and 19 protic solvent to form additional hydrogen gas , which after complexation with Ni2B can reduce the target nitrile function.

8 NaBH4 + NiCl2 + 18 MeOH 2Ni2B + 6 B(OMe)3 + 8 NaCl + 25 H2

etc. 8 NaBH4 + MeOH NaB(OMe)H3 + H2

The formed amine after reduction of the nitrile group is protected in situ by reaction with di-tert- butyl dicarbonate in order to avoid the inherent instability of amino esters. A disadvantage, however is the partial reduction (14% on LCMS) of the ester to the primary alcohol. Moreover, the reducing agent appears to be strong enough to reduce a fraction of the aromatic bromide (up to 11%). This only became clear to us after the subsequent saponification step, since both products at this stage showed the same Rf value on TLC. Application of the same procedure on different acrylates shows the conversion of 10-15% of the ester to the corresponding primary alcohol.

Part 2: Results and discussion

Starting from isobutyl-substituted methyl cyanoacetate VI.42; we also tried reduction methodologies using Pd/C and LiAlH4. Both led to a complex mix of compounds in addition to unreduced VI.42, in contrast to the NaBH4/NiCl2 method which provides the product VI.49 in good yield.

O BocHN BocHN BocHN BocHN COOMe COOMe COOMe COOMe

VI.46 VI.47 VI.48 VI.49

58% 40% 57% 75%

Figure V.8: Yields obtained after reduction of the corresponding acryl compounds and in situ Boc protection.

V.2.2.5 Hydrolysis of the methyl ester:

The hydrolysis of the methyl ester in model intermediate VI.44 to the carboxylic acid proceeds without problems with the aid of NaOH (scheme VI.11). Dioxane has to be added as a co-solvent due to the poor solubility of the methyl ester in H2O/MeOH.

Br Br

2 eq NaOH, H2O/MeOH/dioxane (2/1/1), BocHN o.n, r.t BocHN COOMe COOH

VI.44 VI.50 65%

Scheme VI.11: Hydrolysis of the methyl ester to the corresponding carboxylic acid.

The same procedure could be used for preparation of VI.51 and VI.52. However, to obtain VI.53 and VI.54, the reaction at room temperature was slow (~60% conversion after 24h). Additionally,

Part 2: Results and discussion heating at 50 °C for 20 minutes accomplished the hydrolysis in 80% and 95% yield respectively. Isolated yields of the corresponding carboxylic acids are shown in figure VI.9.

O BocHN BocHN BocHN BocHN COOH COOH COOH COOH

VI.51 VI.52 VI.53 VI.54

84% 78% 80% 95%

Figure VI.9: Additionally prepared N-Boc-β-aminocarboxylic acids via basic hydrolysis.

VI.2.2.6 Boc-removal

The removal of the Boc group was carried out under strongly acidic conditions (HCl), smoothly resulting in 99% yield of VI.55, which could be used in the next step without intermediate purification (scheme VI.12).

Br Br Conc. HCl/dioxane 1/9, 7 h, r.t

BocHN HCl.H2N COOH COOH

VI.50 VI.55 99%

Scheme V.12: Boc-removal of the carboxylic acid using mixture of HCl/dioxane.

Treatment of other Boc-protected β-amino acids, (VI.51, VI.53 and VI.54) under the same conditions, led to the formation of the corresponding ammonium salts in excellent yield (figure VI.10). However, attempted Boc-removal of furyl containing intermediate VI.52 did not yield the desired compound, but led to decomposition and a complex reaction mixture. Further research is

100

Part 2: Results and discussion needed to optimize this step, possibly with another Boc-removal procedure or performing an alternative N-protection during nitrile reduction.

O HCl.H2N HCl.H2N HCl.H2N COOH COOH HCl.H2N COOH COOH

VI.56 VI.57 VI.58 VI.59

99% 99% 99% complex mixture

Figure VI.10: Yields of amine deprotection to the corresponding HCl salts.

VI.2.2.7 Fmoc-protection

Final protection of the obtained model crude ammonium salt VI.55 to the Fmoc-protected-β-amino acid VI.60 is achieved using Fmoc-OSu in the presence of Na2CO3. The protection occurs smoothly overnight and the final product is obtained after final purification by recrystallization, scheme (VI.13).

Br Br

1 eq Fmoc-OSu, 3 eq Na2CO3, THF/H2O, + HCl H N r.t, o.n FmocHN FmocHN . 2 COOH COOH COOH

VI.55 VI.60 VI.61 82%

(VI.60/VI.61: 89/11 ratio)

Scheme VI.13: Fmoc protection of the HCl salt of amino carboxylic acid VI.55.

Further analysis, however, reveals that the product is contaminated with the corresponding benzyl analogue (11%) VI.61 arising from the nitrile reduction step (as mentioned above). Despite multiple attempts, these products turn out to be inseparable (column chromatography / HPLC / crystallization), so it was decided not to use this building block in the solid phase synthesis. Other obtained N-Fmoc -β3-alkyl-amino acids and their yields are shown in figure VI.11.

101

Part 2: Results and discussion

FmocHN FmocHN FmocHN COOH COOH COOH

VI.62 VI.63 VI.64

85% 92% 86%

Figure VI.11: Yields of the N-Fmoc-β3-alkyl-amino acid VI.62-VI.64 after recrystallization

VI.3 SOLID PHASE SYNTHESIS OF 3-SUBSTITUTED-1,5-BENZODIAZOCINE-2,6- DIONES

VI.3.1 Building blocks used

A- Amino acid:

As discussed in chapter IV, our choice of β2-amino acids for library synthesis are Fmoc-protected derivatives VI.62-VI.64, (figure VI.12). These building blocks will determine the nature of the substitution at the C-3 position of the target benzodiazocinediones.

O O O HO NHFmoc HO NHFmoc HO NHFmoc

Cl VI.62 VI.63 VI.64

Figure VI.12 N-Fmoc-β2-amino acids used to synthesize a library of 3-substituted benzodiazocinediones.

B- Alcohols

Introduction of diversity into the 5-position in the benzodiazocinedione skeleton is achieved by a Mitsunobu-Fukuyama reaction sequence. As the actual alkylation step involves a nucleophilic

102

Part 2: Results and discussion substitution (SN2) reaction, (scheme VI.14), the rate of reaction is related to the steric and electronic properties of the intermediate alkoxyphosphonium salt (VI.68). Before selection of suitable alcohols for library construction, a series of primary alcohols were tested (table VI.7). To illustrate reactivity differences, these test reactions were stopped after one hour reaction times. Longer reaction times and / or multiple treatments could lead to better conversions if necessary. The Mitsunobu-Fukuyama alkylation with the aid of secondary alcohols is known to be difficult to happen20,21, so we prefered not to include these secondary alcohols in our libraries. The use of simple primary alkyl alcohols (MeOH, EtOH, pentanol, isopentanol) gives generally very good and fast conversion9. Similarly, methoxyethoxyethanol and 2-aminoethanol derivatives (experiment 2-4) gave good results; the reaction with 3-butyn-1-ol (experiment 5) on the other hand displayed a clear lower reaction rate, while the use of 2,2,2-trifluoroethanol did not result in any product formation. We suggest lowered nucleophilicity may be hampering the formation of the alkylating species VI.68.

O NO O O 2 O O O NO2 S S O N 10 eq alcohol, 5 eq Ph3P, O N 5 eq DIAD, DCE, rt, 1h H R VI.65 VI.66

Via: Ph Ph Ph P O O O NO2 N NH PPh3 S O O O N OH O O O H R R H

SN2

VI.67 VI.68 VI.69

Scheme VI.14: Test of the Fukuyama-Mitsunobu alkylation of VI.65 with different alcohols (see table VI.7).

103

Part 2: Results and discussion

Electronic and structural effects also play a major role in the alkylation with (substituted) benzylic type alcohols. “Neutral” benzylic alcohols (experiments 7 and 8) show excellent reactivity, while cinnamyl alcohol (experiment 9) gives somewhat reduced levels of conversion. Electron poor benzylic substituents (experiments 10 and 11, -I effect of para-F and meta-OMe, respectively) are converted completely, while the presence of mesomerically electron donating substituents (OMe and OCH2Ph, experiments 12-14) in o/p- position led to zero conversion. In line of this, the presence of N(Me)2 in p-position would be expected to be detrimenal for reactivity, but surprisingly (in experiment 15) it shows excellent results.

Table VI.7: Results for the Mitsunobu-Fukuyama alkylation with primary alcohols.

Exp. Alcohol VI.65/VI.66(a) Purity(b) 1 MeOH 0/100 98% 2 O OH 6/94 6% O 3 OH 4/96 92% O N

4 OH 1/99 94% N

5 62/38 90% OH 6 F 100/0 94% F OH F 7 OH 2/98 93%

8 1/99 83% OH

9 OH 28/73 80%

10 OH 1/99 91% F

104

Part 2: Results and discussion

11 OH 2/98 79%

OMe

12 OH 100/0 70% MeO 13 100/0 40% O OH 14 OH 100/0 80%

MeO OMe 15 Me 10/90 94% N Me OH (a) Relative ratio determined from the peak areas in the HPLC chromatogram at 214 nm of the corresponding compounds after TFA cleavage; (b) Total HPLC purity of start and end product in the crude cleavage mixture TFA (214 nm).

Based on these test results, we have chosen to include the alcohols VI.70-VI.80 in a library (figure VI.13). To ensure full conversion in the Mitsunobu reaction, it is decided to run the reaction three times (3 x 2h).

OH MeOH EtOH O OH O VI.70 VI.71 VI.72 VI.73

OH OH OH OH

VI.74 VI.75 VI.76 VI.77 O OH OH OH N

OMe O VI.78 VI.79 VI.80

Figure VI.13 Sets of alcohols chosen for library construction.

105

Part 2: Results and discussion

C- N-Fmoc-anthranilic acids

The different anthranilic acids for the intended library are displayed in figure VI.14 and are chosen for their different aromatic electron density and potential for further derivatization (Br: Suzuki- arylation).

NHFmoc NHFmoc NHFmoc

COOH Br COOH MeO COOH

VI.7 VI.8 VI.9 NHFmoc NHFmoc

COOH Me COOH F VI.10 VI.11

Figure VI.14 Different N-Fmoc anthranilic acids used in the library.

VI.3.2 Library Synthesis

Based on the earlier results from the solid-phase synthetic strategy with ring closure in solution for model compound IV.34 and the above-mentioned Mitsunobu test reactions, we wanted to synthesize a library of 3-substituted-1,5-benzodiazocine-2,6-diones. The experimentally carried synthesis route with detailed reaction conditions is depicted in scheme VI.15.

In a first step, Wang resin was loaded with the Fmoc-protected β2-amino acids. The loading values were determined using Fmoc UV-quantitation and were used for calculation of the overall yield of open-chain compounds VI.88. From these batches, the library synthesis was manually carried out in parallel, using custom-made glass solid-phase vessels, starting from 0.4788 mmol VI.81. Individual reactions were not monitored during the library synthesis, and all steps were executed under the optimized conditions mentioned above.

106

Part 2: Results and discussion

O O O a b c o-Ns OH O NHFmoc O NH O N 2 H R1 R1 R1

V.1 VI.81 VI.82 VI.83

O O O O NHFmoc d o-Ns e f O N O NH O N

R1 R2 R1 R2 R1 R2 R3

VI.84 VI.85 VI.86

H O O O NH O O NH2 2 N R b g h 1 O N HO N R3 R1 R2 R1 R2 N R R 3 3 O R2 VI.87 VI.88 VI.1

Reagents and Conditions: a)1) 2eq VI.62-VI.64, 2 eq DIC, CH2Cl2, 0.2 eq DMAP, 24h. 2) Ac2O/ DIPEA/DCM 1/1/3 , 2x2 h. b) 20% 4-methylpiperidine in DMF 2 x 20 min. c) 5eq o-NsCl, 10eq collidine, CH2Cl2, 2x1h .d) 10eq VI.70- VI.80, 5eq PPh3, 5eq DIAD, DCE, 3x2h. e) 2.5 eq DBU, 5eq 2-mercaptoethanol, DMF, 2x30 min. f) 10eq Fmoc- anthranilic acid VI.7-VI.11, 5eq DIC, DCM/DMF 9/1, 24 h. g) TFA/H2O 95/5, 2x1h. h) polystyrene-bound-DCC, DCM, 1h. Scheme VI.15: Synthetic route with detailed reactions conditions to synthesize 3-substituted-1,5- benzodiazocine-2,6-dione

An overview of the synthesized compounds as well as a list of accompanying solid phase synthesis yields and cyclization yields is shown in table VI.8 and figure VI.16. A library of 13 compounds were thus prepared and purified using column chromatography (HPLC were needed in some cases).

107

Part 2: Results and discussion

Table VI.8: Solid-phase synthesis (SPS) and solution cyclization (CYC) yields of the synthesized library of 3-substituted-1,5-benzodiazocine-2,6-diones.

[a] [b] Product R1 R2 R3 SPS% CYC% VI.89 i-Bu Me H 65 67

VI.90 i-Bu CH2CH2OCH2CH2OCH3 7-Flouro 53 55 VI.91 i-Bu 4-Phenyl benzyl 8-Methoxy 31 30 VI.92 i-Bu 3-Methoxybenzyl H 62 41 VI.93 i-Bu Cinnamyl H 38 44 VI.94 2-naphthylmethyl Me H 37 80 VI.95 2-naphthylmethyl Ethyl 8-Bromo 63 63 VI.96 2-naphthylmethyl 3-But-1-ynyl 8-Methoxy 35 21 VI.97 2-naphthylmethyl 4-Fluorobenzyl 8-Methyl 62 41 VI.98 p-chlorobenzyl Methyl 8-Methoxy 87 25 VI.99 p-chlorobenzyl i-Pentyl H 69 39 VI.100 p-chlorobenzyl 2-(2,5-dioxopyrrolidin-1- H 30 43 yl)ethyl VI.101 p-chlorobenzyl Benzyl 8-Bromo 50 27 [a] Isolated yields are determined after purification and based upon the initial resin loading. [b] Isolated yields.

median

SPS % 30 31 35 37 38 50 53 62 62 63 65 69 87 average=52

CYC % 21 25 27 30 39 41 41 43 44 55 63 67 80 average=44

Figure VI.16: Yields of solid-phase synthesis and cyclization.

108

Part 2: Results and discussion

O O H O H H N N N

N N MeO N O F O O VI.90 O VI.89 O VI.91 SPS: 65% SPS% 53% SPS : 31% CYC: 67% CYC: 55% CYC: 30 % O O H O H H N N N

N N N O O O

VI.92 VI.93 VI.94 SPS: 62% OMe SPS 38% SPS: 37% CYC: 41% CYC: 44% CYC: 80% O O H H H O N N N

Br N MeO N Me N O O O

VI.95 VI.96 VI.97 F SPPS: 63% SPS 35 % SPPS: 62% CYC: 63% CYC: 21% CYC: 41% Cl Cl Cl Cl

O O O H O H H N H N N N

Br N Br MeO N N N O O O O O N VI.98 VI.99 VI.100 O VI.101 SPS: 50% SPS: 69% SPS: 30% SPS: 87% CYC:27% CYC: 39% CYC: 43% CYC:25%

Figure VI.17: Synthesized library of 3-substituted-1,5-benzodiazocine-2,6-diones.

Yields of the solid-phase synthesis of α,ω-amino carboxylic acids VI.88 range between 30% and 87% ( median: 53%, average: 52%), while cyclization yields vary from 21% to 80% (median: 41%, average: 44%). No particular substituent effects seem to be responsible for these fluctuations.

VI.3.3 Conclusion

In conclusion, the earlier established conditions could be applied to a library of 3-substituted-1,5- benzodiazocine-2,6-diones. It seems the varying yields of cyclization are a consequence of varying

109

Part 2: Results and discussion amounts of N-acylating resin-bound side products, as the reaction mixtures are generally very clean. This has thus to be interpreted as resulting from an increased susceptibility to this unwanted N→O acyl migration reaction or could be a consequence of slower rates of cyclization.

It may be therefore suggested to repeat those cyclizations under the alternative ClCOOMe-induced conditions as described for the model benzodiazocinedione IV.34 (chapter V)

110

Part 2: Results and discussion

VI.4 CONCLUSION

Based on the proposed solid phase synthesis strategy for the 3-substituted-1,5-benzodiazocine-2,6- diones VI.1, essential building blocks consist of N-Fmoc-anthranilic acids and N-Fmoc-2-alkyl-3- aminopropionic acids. The former could be accessed by a one-step synthesis from corresponding commercially available 2-aminobenzoic acids. Fmoc protection with Fmoc-OSu delivered five N- Fmoc-2-aminobenzoic acid derivatives VI.7-VI.11 (57-85%).

3 3 NHFmoc 2 NH2 4 2 4 i) 1 eq NaOH, H2O, rt, 5 min R R 1 1 5 5 ii) 1 eq NaHCO3, 1 eq Fmoc-OSu, COOH 6 COOH 6 THF/H2O 2/1, r.t, o.n

2-aminobenzoicacid Fmoc-2-aminobenzoic acid

R= H R= H VI.7 R=5-bromo R=5-bromo VI.8 R=5-methoxy R=5-methoxy VI.9 R=5-methyl R=5-methyl VI.10 R=6-fluoro R=6-fluoro VI.11

NHFmoc NHFmoc NHFmoc

COOH Br COOH MeO COOH

VI.7 VI.8 VI.9 NHFmoc NHFmoc

COOH Me COOH F VI.10 VI.11

Scheme VI.16 Synthesis of N-Fmoc-2-aminobenzoic acid derivatives.

N-Fmoc-2-alkyl-3-aminopropionic acid building blocks were successfully prepared starting from the appropriate aldehyde and methyl cyanoacetate which were reacted via a Knoevenagel condensation followed by reduction using Pd/C-catalyzed hydrogenation (R=isopropyl) or

NaCNBH3/AcOH to give compounds VI.104. Chemoselective reduction of the cyano group using 2 NiCl2.6H2O/NaBH4 and in situ Boc-protection gave protected β -amino acid intermediates VI.105. Hydrolysis of the ester moiety in those compounds was achieved using NaOH, while the N-

111

Part 2: Results and discussion terminus was reprotected as fluorenylmethyl carbamate to the Fmoc-protected amino acids using

Fmoc-OSu/Na2CO3. The building blocks were obtained in good overall yields.

g (R= isopropyl)

O a Boc COOMe d NC COOMe b NC COOMe c N H H R (R=2-naphthyl or R 4-chlorophenyl R R VI.102 VI.103 VI.104 VI.105

Boc COOH e COOH f Fmoc COOH N HCl.H2N N H H R R overall yield R VI.64: R = isopropyl 55%. VI.106 VI.107 VI.63: R = 2-naphthyl 34%. VI.62: R = 4-chlorophenyl 50%.

Reagents and conditions: a) methyl cyanoacetate, AcOH, piperidine, dioxane, r.t., o.n. b) NaCNBH3, AcOH, MeOH, r.t, 1h to o.n. c) NiCl2.6H2O, NaBH4, Boc2O, MeOH, 0C to r.t., o.n. d) NaOH, H2O, MeOH, r.t to 50° C, 20 min to o.n. e) Dioxane / concentrated HCl 9/1, r.t, 7h to o.n. f) Na2CO3, Fmoc-OSu, THF/H2O (2/1), r.t., o.n. g) methyl cyanoacetate, AcOH, piperidine, dioxane, Pd/C 5% wt, H2 (2 bar), r.t, 2 h. Scheme VI.17 Synthesis of N-Fmoc-2-alkyl-3-aminopropionic acid.

COOH COOH FmocHN FmocHN

O Br VI.108 VI.22

Figure VI.18 Intended compounds which could not be obtained.

Intended compounds VI.108 and VI.22 could not be obtained successfully as bromobenzyl derivative VI.22 was contaminated with a substantial amount (11%) of the de-halogenated analogue, while for VI.108, the final N-protection sequence could not be achieved because of total degradation in the acidic medium used for Boc-removal.

Alcohols VI.70-VI.80 were chosen to decorate the N-5 position in the target 3-substituted-1,5- benzodiazocine-2,6-diones.

112

Part 2: Results and discussion

OH MeOH EtOH O OH O VI.70 VI.71 VI.72 VI.73

OH OH OH OH

VI.74 VI.75 VI.76 VI.77 O OH OH OH N

OMe O VI.78 VI.79 VI.80

Figure VI.19 Sets of alcohols chosen for library construction.

The synthetic route towards 3-substituted-1,5-benzodiazocine-2,6-dione ran parallel to model 1,5- benzodiazocine-2,6-dione, applying the N-Fmoc-β2-amino acids in the proposed synthetic route. The overall yields of solid-phase synthesis of ring-closing precursors are moderate to good (30- 87%, median 53%). Cyclization yields using polystyrene-bound DCC were low to moderate (21- 80%, median 41%), in agreement with expected O→N acyl migration side reactions.

113

Part 2: Results and discussion

O H H O N N

8 N R R2 7 2 N O R 1 O R1 VI.89 R1=methyl, R2=H VI.94 R1= methyl, R2= H VI.90 R1=2-(2-methoxyethoxy)ethyl, R2=7-fluoro VI.95 R1= ethyl, R2= bromo VI.91 R1=4-phenylbenzyl, R2=8-methoxy VI.96 R1=3-Buty-1-ynyl, R2=methoxy VI.97 R =4-fluorobenzyl, R =methyl VI.92 R1=3-methoxybenzyl, R2=H Cl 1 2 VI.93 R1=cinnamyl, R2=H

H O N

R2 N

O R1

VI.98 R1=methyl, R2=methoxy VI.99 R1=i-pentyl, R2=H VI.100 R1= 2-(2,5-dioxopyrolidin-1-yl)ethyl, R2=H VI.101 R1=benzyl, R2=bromo

Figure VI.17: Synthesized library of 3-substituted-1,5-benzodiazocine-2,6-diones.

114

Part 2: Results and discussion

REFRENCES

1 Organic syntheses, Coll. Vol. 1, p.327 (1941); Vol. 5, p. 71 (1925).

2 Gerard, A.; Lisowski, V.; Rault, S. Tetrahedron 2005, 61, 6082-6087.

3 Liu, Y.; Ye, C.; Chen, Q.; Yang, G. Tetrahedron Lett. 2013, 54, 949-955.

4 Dawei, M.; Qingquan, W. Tetrahedron Lett. 2001, 42, 5279-5281.

5 Beak, P.; Snieckus, V. Acc. Chem. Res. 1982, 15 (10), 306–312.

6 Muchowski, J. M.; Venuti, M. C. J. Org. Chem. 1980, 45, 4798-4801.

7 Gould, S. J.; Eisenberg, R. L. J. Org. Chem. 1991, 56, 6666-6671.

8 Seebach, D.; Wasmuth, D. Helv. Chem. Acta 1980, 63(17), 197-200.

9 Caroen J., Ontwikkeling van een vastefasesynthesestrategie voor 1,2,3,4,5,6-hexahydro-1,5- benzodiazocine-2,6-dionen voor toepassing in combinatorische bibliotheken, (Development of a solid- phase synthesis strategy for 1,2,3,4,5,6-hexahydro-1,5-benzodiazocine-2,6-diones for application in combinatorial libraries) 2012. PhD dissertation, UGent. 10 Kaptein, B.; Broxterman, Q.; Kierkels, J.; Gerardus, T.; Milcent, T., WO Patent 2,005,085,462: 2005.

11 A few alternative procedure are described in literature, using Ni, PtO2 or Pd(OH)2, but we have not investigated these. Ni: Ghislieri, D.; Gilmore, K.; Seeberger, H. Angew. Chem. Int. Ed. 2015, 45, 678-682;

PtO2: Menichincheri, M.; Bargiotti, A.; Berthelsen J.; Bertrand J. A.; Bossi R.; Ciavolella A.; Cirla A.; Cristiani C.; Croci V.; D’Alessio R.; Fasolini M.; Fiorentini F.; Forte B.; Isacchi A.; Martina K.; Molinari A.; Montagnoli A.; Orsini P.; Orzi F.; Pesenti E.; Pezzetta D.; Pillan A.; Poggesi I.; Roletto F.; Scolaro A.; Tatò M.; Tibolla M.; Valsasina B.; Varasi M.; Volpi D.; C. Santocanale; Vanotti E. J. Med.

Chem., 2009, 52(2), 293–307; Pd(OH)2: Lee, J.; Gauthier D.; Rivero, R. A. J. Org. Chem., 1999, 64, 3060- 3065. And Peukert, S.; Sun, Y.; Zhang, R.; Hurley, B.; Sabio, M.; Shen, X.; Gray, C.; Dzink-Fox, J.; Toa, J.; Cebula, R.; Wattanasin S. Bioorg. Med. Chem. Lett. 2008, 18, 1840-1844.

12 Hayashi, T. J. Org. Chem., 1966, 13(10), 3253-3258.

13 Russo, A. T.; Amezcua, K. L.; Huynh, V. A.; Rousslang, Z. M.; Cordes, D. B. Tetrahedron Lett. 2011, 52(50), 6823-6826.

14 Hutchins, R. O.; Rotstein, D.; Natale, N.; Fanelli, J.; Dimmel, D. J. Org. Chem. 1976, 41(20), 3328- 3329.

15 Nystrom, R. F.; Brown, W. G. J. Am. Chem. Soc. 1947, 69(5), 1197-1199.

16 Badorrey, R.; Cativiela, C.; Dı́az-de-Villegas, M. a. D.; Gálvez, J. A.; Gil, A. Tetrahedron: Asymmetry 2003, 14(15), 2209-2214.

17 Lu, Y.; Miet, C.; Kunesch, N.; Poisson, J. E. Tetrahedron: Asymmetry 1993, 4(5), 893-902.

18 Caddick, S.; Judd, D. B.; Lewis, A. K. d. K.; Reich, M. T.; Williams, M. R. Tetrahedron 2003, 59(29), 5417-5423.

115

Part 2: Results and discussion

19 Brown, C. A.; Brown, H. C. J. Am. Chem. Soc. 1963, 85(7), 1003-1005.

20 Olsen, C. A.; Witt, M.; Hansen, S. H.; Jaroszewski, J. W.; Franzyk, H. Tetrahedron 2005, 61, 6046-

6055. The optimized conditions according to these authors are: 6 eq Ph3P, 5 eq DEAD, 5 eq alcohol (5 eq = 0.25M), dichloromethane / THF 1/1, r.t, 2 x 3h.

21 Olsen, C. A.; Jorgensen, M. R.; Witt, M.; Mellor, I. R.; Usherwood, P. N. R.; Jaroszewski, J. W.; Franzyk, H. Eur. J. Org. Chem. 2003, 3288-3299. The optimized conditions (in solution) are as follows: 1.5 + 1 eq Me3P, 1.5 eq 1 + 1,1 '( azodicarbonyl) dipiperidide (ADDP), 1.5 eq of alcohol (1.5 eq = 0.1 M), toluene / THF 8/1, rt, 22 h + 22 h.

116

Part 2: Results and discussion

VII SYNTHESIS OF 4-SUBSTITUTED-1,5-BENZODIAZOCINE-2,6-DIONES

Our second target library consists of the 4-substituted-1,5-benzodiazocine-2,6-diones VII.1. To obtain these 8-membered bis-lactams, we expect that above-used solid-phase synthesis/solution- phase cyclization strategy could be applied here as well.

VII.1 RETROSYNTHESIS

The 4-substituted-1,5-benzodiazocine-2,6-diones VII.1 are structurally similar to their 3- substituted counterparts described in chapter VI and should therefore be accessible in a similar synthetic fashion (figure VII.1). The final step consists of the ring closure through lactam bond formation. Subsequently, the second amide bond in VII.2 can be disconnected, resulting in anthranilic acid building blocks VII.4 and solid-supported β3-amino acids (VII.3) which can be synthesized from simple coupling of N-Fmoc-3-amino-3-alkyl propionic acids VII.5 to solid support VII.6.

NHFmoc HOOC

R3 anthranilic acids VII.4 H O 10 amide bond + N Lactam 9 1 2 O R1 O NH2 formation O R1 3 formation R3 4 8 6 5 O N O NH N R1 7 R R O R 2 2 2 R3 VII.1 VII.2 VII.3

3-amino acid coupling

O R1 OH + HO NHFmoc 3-amino acid VII.6 VII.5

Figure VII.1: Retrosynthetic overview of the approach towards 4-substituted-1,5-benzodiazocine- 2,6-diones VII.1.

117

Part 2: Results and discussion

VII.2 BUILDING BLOCK SYNTHESES

VII.2.1 Synthesis of N-Fmoc-anthranilic acid derivatives

Besides the anthranilic acids VI.7-VI.10 in chapter VI; we additionally prepared N-Fmoc- protected 2-amino-4-trifluoromethylbenzoic acid VII.7 (yield: 62%). These five building blocks were used for the library synthesis.

NHFmoc NHFmoc NHFmoc

COOH Br COOH MeO COOH

VI.7 VI.8 VI.9 F3C NHFmoc NHFmoc

COOH Me COOH V.10 VII.7

Figure VII.2: N-Fmoc-2-aminobenzoic acid derivatives

VII.2.2 Synthesis of N-Fmoc-3-alkyl-3-aminopropionic acid building blocks:

Essential building blocks for the synthesis of 4-substituted benzodiazocinediones using our strategy, are N-Fmoc-3-alkyl-3-aminopropionic acids VII.5. For a first library we choose derivatives VII.9-VII.11, which would lead to 3-methyl-, 3-isobutyl-, and 3-benzyl-substituted 1,5-benzodiazocine-2,6-diones. These building block were synthesized from the corresponding commercially available unprotected β3-amino acids VII.8, and are accessed by treatment of the latter with equivalent amounts of 9-fluorenylmethyl-N-succinimidyl carbonate (Fmoc-OSu) (scheme VII.1).

118

Part 2: Results and discussion

O R O R 2 eq Na CO , 1 eq Fmoc-OSu, 2 3 HO NHFmoc HO NH2 THF/H2O 2/1, r.t, o.n VII.8 VII.5

O O O

HO NHFmoc HO NHFmoc HO NHFmoc

VII.9 VII.10 VII.11

Scheme VII.1: Synthesis of N-Fmoc-3-alkyl-3-aminopropionic acid derivatives.

Table VII.1: Yields of the prepared N-Fmoc-3-amino-3-alkyl propionic acid derivatives

Entry Compound R Yielda

1 VII.9 Me 90 % 2 VII.10 i-Bu 87 % 3 VII.11 Bn 84 % a Yields are calculated after purification

VII.3 SOLID PHASE SYNTHESIS OF 4-SUBSTITUTED-1,5-BENZODIAZOCINE-2,6- DIONES

After the successful synthesis of 3-substituted-1,5-benzodiazocine-2,6-diones, we followed the same solid-phase synthetic strategy to attempt synthesis of the 4-substituted analogues.

VII.3.1 Coupling of Fmoc-β-amino acid on Wang resin and Fmoc removal

In a first stage, the coupling of three racemic β3-amino acid derivatives (VII.9-VII.1) was performed. Wang resin was treated with DIC-activated amino acid in the presence of DMAP, followed by a capping procedure. The efficiency of the coupling step (table VII.2) was determined via a quantitative UV-based Fmoc determination method upon extensive drying of the beads.

119

Part 2: Results and discussion

1. a) 2 eq Fmoc-AA-OH 2 eq DIC 20 min, CH2Cl2, 0°C b) 0.2 eq DMAP O R1 CH Cl , r.t, 24h OH 2 2 O NHFmoc

2. Ac2O/DIPEA/CH2Cl2 Wang (1/1/3), r.t, 2 x 1h VII.6 VII.12

O O O

O NHFmoc O NHFmoc O NHFmoc

VII.13 VII.14 VII.15

Scheme VII.2: Coupling of N-Fmoc-3-amino-3-alkylpropionic acid VII.9–VII.11 to Wang resin.

Table VII.2: Coupling efficiency of loading amino acids to Wang resin.

Entry Compound loadinga 1 VII.13 0.67 mmol/g ± 0.01 2 VII.14 0.69 mmol/g ± 0.01 3 VII.15 0.64 mmol/g ± 0.01 a determined by Fmoc-UV-quantitation

Fmoc removal occurred with 4-methylpiperidine in DMF delivering the resin-bound primary amine.

O R1 O R1 O NHFmoc 20% 4-methylpiperidine in DMF, 2 x 20 min, rt O NH2 VII.12 VII.16

Scheme VII.3: Fmoc removal using a solution of 20% 4-methylpiperidine in DMF.

120

Part 2: Results and discussion

VII.3.2 Mitsunobu-Fukuyama reaction sequence

To achieve N-mono alkylation, a Mitsunobu-Fukuyama alkylation sequence was performed. To this end, the resin-bound primary amine was initially treated with 2-nitrobenzenesulfonyl chloride (o-NsCl) in the presence of collidine (2,4,6-trimethylpyridine). At this stage, the actual Mitsunobu alkylation takes place with alcohols R2OH (10 eq) in the presence of triphenylphosphine (Ph3P, 5 eq, 0.2 M) and diisopropyl azodicarboxylate (DIAD, 5 eq, 0.2 M). For this library, we have chosen to include the alcohols VII.20-VI.26 to decorate the N-5 position (figure VII.3, VII.22 was prepared via Cbz-protection of 4-aminobutanol). To ensure complete alkylation, this step was repeated two more times. Deprotection to resin-bound secondary amines VII.19 occurs with the aid of β-mercaptoethanol and DBU (scheme VII.4). LCMS analysis shows the unique formation of the expected products.

o-Ns

O R 1 O R1 O O NO2 5 eq o-Ns-Cl, 10 eq collidine S O NH2 O N CH Cl , r.t, 2x1h 2 2 H

VII.16 VII.17

10 eq R2OH, 5 eq Ph3P, 5 eq DIAD, DCE, r.t, 3x2 h

O R1 O R1 O O NO2 OH S O NH 5 eq HS , 2.5 eq DBU O N R2 R2 DMF, r.t, 2x30 min VII.19 VII.18

Scheme VII.4: Mitsunobu-Fukuyama alkylation three step reaction sequence, applied to resin- bound β-amino acid.

121

Part 2: Results and discussion

O MeOH OH OH O N H

VII.20 VII.21 VII.22

OH OH OH OH MeO F

CF3 VII.23 VI.24 VII.25 VII.26

1 eq Et N, 1 eq PhCH OCOCl, HO HO 3 2 NHCbz NH2 0.1 M CH2Cl2, 0C to r.t., 6 h 98% VII.27 VII.22

Figure VII.3 Set of alcohols chosen for library construction.

VII.3.3. Coupling of Fmoc-anthranilic acid derivatives

Due to the presence of proximal substituents in the β3-position of the resin-bound secondary amino acid, we can expect that steric factors could play an important role in the coupling of Fmoc- anthranilic acid building blocks. Using VII.28 as a model substrate, we found it was indeed necessary to repeat the coupling step (2 x 24h) procedure as used for the resin-bound β2-substituted amino acids. In this way, VII.30 could be isolated in 48% yield compared to 33% yield after single treatment with (Fmoc-Abz)2O. As a consequence, for the rest of the library this amide forming step was always repeated once.

122

Part 2: Results and discussion

O R1 O R1 O NHFmoc O NH O N 10eq VII.4 5 eq DIC, CH Cl /DMF 9/1, R2 2 2 R 0C to r.t. 2x24 h 2 R VII.19 VII.29 3

R =R =Bn, VII.28 (model compound) R1=R2=Bn, R3=H, VII.30, 1 2 (model compound)

(i) 20% 4-methylpiperidine in DMF, 2 x 20 min (ii) TFA/H2O 95/5, 1 h

O Bn O NH2 HO N Bn

VII.31 48% from VII.15

Scheme VII.5 Coupling of anthranilic acid anhydride to resin-bound amines VII.19.

VII.3.4. Fmoc removal and cleavage from resin

The removal of the fluorenylmethyloxycarbonyl moiety occurs smoothly by treating VII.29 with a solution of 20% 4-methylpiperidine in DMF 2x20 min, which is immediately followed by cleavage of the dipeptides VII.33 from the resin by shaking in a solution of TFA/H2O 95/5 for 1 hour. These products were purified using column chromatography before final cyclization.

123

Part 2: Results and discussion

O R1 O NHFmoc O R1 O NH2 O N O N 20 % 4-methylpiperidine in DMF R2 R2 R3 2x 20 min R3 VII.29 VII.32

TFA/H2O 95/5 1 h

O R1 O NH2 HO N

R2 41-95% R3 VII.33

Scheme VII.6 Fmoc deprotection followed by TFA-cleavage from the resin.

In this way, a library of 11 compounds was successfully obtained. Isolated yields varied between 41% and 95% (calculated from VII.12, average yield: 60 %; median yield: 56%, table VII.3).

VII.3.5. Cyclization using DCC-resin bound

From the purified intermediates VII.32 , application of the ring closing conditions as described before (DCC-PS, 1h) readily afford the desired 4-substituted-1,5-benzodiazocine-2,6-diones VII.1 in yields between 18% and 65% ( average yield: 40%, median yield: 45%).

Again, the crude reaction mixtures show clean formation of the expected eight-membered final products without remaining starting material. The low to moderate yields are interpreted to arise from O→N acyl migration of the resin-bound O-acylisourea activated species, as discussed before for the 3-substituted analogues.

124

Part 2: Results and discussion

O O R O NH H 1 2 N HO N 3 eq DCC-PS, 0.01 M DCM, R3 R 2 1 h, r.t N R1 R3 O R2 18-65% VII.33 VII.1

Scheme VII.7 Cyclization of the ring closed precursor using DCC-resin.

However, no obvious correlation of observed yields to the corresponding structures could be established, although the presence of p-bromo and p-methoxy groups on the anthranilamine moiety seems to lower the yields of cyclization.

Table VII.3: Overview of the synthesized 4-substituted-1,5-benzodiazocine-2,6-diones.

[a] [b] Product R1 R2 R3 SPS% Cyc% VII.34 Me Me H 77 65 VII.35 Me Bn H 47 45 VII.36 Me 3-(trifluoromethyl)benzyl 8-Methoxy 57 23 VII.37 Me 4-N(Cbz)butyl 8-Methoxy 41 18 VII.38 Bn Me H 76 46 VII.39 Bn Bn H 48 40 VII.40 Bn Pentyl 8-Bromo 44 31 VII.41 Bn 2-Methoxyethyl 9-Trifluoromethyl 56 57 VII.42 Bn p-Fluorobenzyl 8-Bromo 56 24 VII.43 i-Bu Me H 98 45 VII.44 i-Bu Bn H 73 45 [a] Isolated solid-phase synthesis yield of VII.33, measured after purification and based upon the initial resin loading of VII.12. [b] Isolated yields of cyclization after purification.

125

Part 2: Results and discussion

median

SPS % 41 44 47 48 55 56 57 70 73 76 98 average=60

CYC % 18 23 24 31 40 45 45 45 46 57 65 average=40

Figure VII.4: Yields of solid-phase synthesis and cyclization.

O O O H H H H O N N N N

MeO N N N MeO N O O O O H O N VII.34 VII.35 VII.36 VII.37 O SPS: 77% SPS: 47% SPS: 57% CF SPS: 41% CYC: 65% CYC: 45% CYC: 23% 3 CYC:18%

O O H O H H N N N

Br N N N O O O VII.38 VII.39 VII.40 SPS: 76% SPS: 48% SPS: 44% CYC: 46% CYC: 40% CYC: 31% O O O H H H O H N N N N F3C

Br N N N N O O O O F VII.41 VII.42 VII.43 VII.44 SPS: 56% OMe SPS: 56% SPS: 98% SPS: 73% CYC: 57% CYC: 24% CYC: 45% CYC: 45% Figure VII.5 Synthesized library of 4-substituted-1,5-benzodiazocine-2,6-diones.

126

Part 2: Results and discussion

VII.4 CONCLUSION

Based on the proposed solid-phase synthesis strategy, essential building blocks consist of N-Fmoc- anthranilic acids and N-Fmoc-3-amino-3-alkylpropionic acids. Both could be accessed by a one- step synthesis from their commercially available counterparts. N-Fmoc-anthranilic acids used in this library are VI.7 to VI.10 in addition to VII.7 (figure VII.6).

NHFmoc NHFmoc NHFmoc

COOH Br COOH MeO COOH

VI.7 VI.8 VI.9 F3C NHFmoc NHFmoc

COOH Me COOH V.10 VII.7

Figure VII.6: N-Fmoc-2-aminobenzoic acid derivatives

N-Fmoc-3-amino-3-alkylpropionic acid building blocks were successfully prepared by treating the commercially available β3-amino acids with equivalent amounts of (Fmoc-OSu) in the presence of Na2CO3.

O R O R 2 eq Na CO , 1 eq Fmoc-OSu, 2 3 HO NHFmoc HO NH2 THF/H2O 2/1, r.t, o.n VII.8 VII.9

O O O

HO NHFmoc HO NHFmoc HO NHFmoc

VII.10 VII.11 VII.12

Scheme VII.8: Synthesis of N-Fmoc-3-alkyl-3-aminopropionic acid derivatives.

127

Part 2: Results and discussion

Alcohols VII.20-VII26 were chosen to decorate the N-5 position in the target 4-substituted-1,5- benzodiazocine-2,6-diones.

O MeOH OH OH O N H

VII.21 VII.22 VII.23

OH OH OH OH MeO F

CF3 VII.24 VI.25 VII.26 VII.27

Figure VII.7 Set of alcohols chosen for library construction.

The synthetic route towards 4-substituted-1,5-benzodiazocine-2,6-dione ran parallel to 3- substituted-1,5-benzodiazocine-2,6-dione derivatives, applying the N-Fmoc-β3-amino acids in the proposed synthetic route. Due to steric reasons, a double coupling procedure of N-Fmoc anthranilic was found to be necessary. The overall moderate to good yields (41-98%, median 56%) for the solid-phase synthesis of ring-closing precursors were comparable to the ones obtained for the 3- substituted-1,5-benzodiazocine-2,6-diones. Cyclization yields using polystyrene-bound DCC were low to moderate (18-65%, median 45%), which are attributed with expected O→N acyl migration side reactions.

128

Part 2: Results and discussion

O H O H N N 9 R2 8 R2 N N

O R1 O R1

VII.38 R = methyl, R = H VII.34 R1=methyl, R2=H 1 2 VII.35 R1=benzyl, R2=H VII.39 R1= benzyl, R2= H VII.36 R1=3-(trifluoromethyl)benzyl, R2=methoxy VII.40 R1=pentyl, R2=8-bromo VII.37 R1=N(Cbz)butyl, R2=methoxy VII.41 R1=2-methoxyethyl, R2=9-trifluoromethyl VII.42 R1=p-fluorobenzyl, R2=8-bromo

H O N

R2 N

O R1

VII.43 R1= methyl, R2= H VII.44 R1= benzyl, R2= H Figure VII.8 Synthesized library of 4-substituted-1,5-benzodiazocine-2,6-diones.

129

Part 2: Results and discussion

VIII. SYNTHESIS OF 3,3-DISUBSTITUTED-1,5-BENZODIAZOCINE-2,6-DIONES

VIII.1 RETROSYNTHESIS

Another goal of this project was to explore the potential synthesis of 3,3-disubstituted-1,5- benzodiazocine-2,6-diones VIII.1 (figure VIII.1). Until now only model analogues with two methyl substituents (VIII.1, R1=R2=Me) have been synthesized at our laboratory. The purpose of this study is to evaluate the possible application to more diverse analogues. The synthetic advantage towards these analogues would be the possibility to perform the cyclization directly from the corresponding (Wang) resin-bound precursors, therefore avoiding the separate TFA- cleavage and solution-phase cyclization procedure. Based on earlier work, this could lead to final products with high crude purity, as unwanted side products would stay resin-bound (see chapter VI.1).

NHFmoc HOOC

R4 anthranilic acids VIII.4

H O + N R1 lactam O O NH2 amide bond O formation formation R4 R 2 O N O NH N R R 1 R R 1 R2 R O R 2 3 3 3 R4 VIII.1 VIII.2 VIII.3

2,2-amino acid coupling and N- alkylation (R3OH)

O + OH HO NHFmoc R 1 R2 VIII.6 VIII.5

Figure VIII.1 Retrosynthesis for 3,3-disubstituted-1,5-benzodiazocine-2,6-diones VIII.1

As before, obviously the disconnection of the 3,3-disubstituted benzodiazocinedione VIII.1 occurs at the N1-C2 lactam bond, similarly to the strategies for 3- and 4-substituted benzodiazocinediones, (figure VIII.1). Subsequently, the second amide bond can be disconnected,

130

Part 2: Results and discussion resulting in anthranilic acid building blocks VIII.4 and a solid-supported β2,2-amino acid (VIII.3) which can be synthesized from coupling of N-Fmoc-3-amino-3,3-dialkylpropionic acid VIII.5 to solid support VIII.6.

VIII.2 BUILDING BLOCK SYNTHESIS

VIII.2.1 Synthesis of N-Fmoc-2,2-dialkyl-3-aminopropionic acid building blocks

Essential building blocks for the synthesis of 3,3-disubstituted-1,5-benzodiazocine-2,6-diones are N-Fmoc-2,2-dialkyl-3-aminopropionic acids. To test the synthesis of 3,3-dialkylaminopropionic acid, our initial choice fell on the use of model building blocks VIII.7 and VIII.8. In the following section our efforts to synthesis these β2,2-(dialkyl)-β-amino acids are described.

H Me H Me N N Fmoc COOH Fmoc COOH

VIII.7 VIII.8

Figure VIII.2 Model compounds β2,2-(dialkyl)-β-amino acid.

VIII.2.1.1 Alkylation of methyl cyanoacetate

Our initial experiments started from VIII.10 (which is prepared from methyl cyanoacetate, see chapter VI) for which the alkylation with methyl iodide towards VIII.11 was tested using different conditions, scheme VIII.1, table VIII.1.

table VIII.1 Me NC COOMe 1 eq isobutyraldehyde, 0.1 eq AcOH, 0.4 eq piperidine, dioxane, 0.1 eq Pd/C 5 wt%, 2 bar, 2 h.NC COOMe NC COOMe

VIII.9 VIII.11 VIII.10

Scheme VIII.1 Alkylation sequence to model intermediate VIII.11.

131

Part 2: Results and discussion

In experiment 1, methylation is tested in DMF using K2CO3 as a base; leading to the desired product VIII.11 in moderate (50%) yield. Changing the solvent to acetone, led to a low yield (10%). Using NaOMe as base with excess MeI or NaH as base, in MeI as solvent, provides the product in 40% and 22% yield respectively. A major practical problem with the isolation of product VIII.11 seems to be its high volatility, hampering the reproducible successful large scale synthesis.

Table VIII.1 Different conditions used for methylation of VIII.10.

Exp. Reagents Solvent T Δt Resultsa

1 1 eq K2CO3 DMF 0 °C to r.t 1 h 50% product 1 eq MeI 0.1 M

2 1 eq K2CO3 Acetone 0 °C to r.t o.n 10% product 1 eq MeI 0.1 M 3 1 eq NaOMe THF 0 °C to r.t o.n 40% Product 2 eq MeI 0.1 M 4 1 eq NaH MeI 0 °C 1h 22% product 0.1 M a Products are obtained after purification with column chromatography

The problem of high volatility of VIII.11 forced us to focus on the synthesis of the second model building blocks VIII.8.

Alkylation of compound VIII.12 with excess methyl iodide in presence of NaOMe, indeed afforded the quaternary product VIII.13 in a very good yield, scheme VIII.2.

i) 1 eq isobutyraldehyde, 0.1 eq AcOH, 0.4 eq piperidine, dioxane, o.n, r.t. 5 eq MeI, 2eq NaOMe, THF 1M, 1h. Me NC COOMe ii) 1.1 eq NaBH CN, 0.4 M AcOH, 3 NC COOMe NC COOMe MeOH/CH2Cl2 (5/1), r.t, 1 h 78% VIII.9 VIII.12 VIII.13

Scheme VIII.2 Alkylation of VIII.12 with methyl iodide in presence of NaOMe.

132

Part 2: Results and discussion

VIII.2.1.2 Reduction of the nitrile group

The earlier mentioned successful reduction of the nitrile group in cyanoacetates (chapter VI) using

NiCl2.6H2O and NaBH4, followed by in situ protection with di-tert-butyl dicarbonate is applied here to obtain the Boc-protected β2,2-amino ester VIII.14 in 60% yield. A partial reduction (20%) of the ester moiety to the primary alcohol VIII.15 could not be avoided (as also described in chapter VI).

+ 2 eq Boc2O, 0.1 eq NiCl2.6H2O, H Me H Me Me N N OH 7 eq NaBH4, 0C to r.t, o.n Boc COOMe Boc NC COOMe

60% 20% VIII.13 VIII.14 VIII.15

Scheme VIII.3 Reduction of the cyano group in VIII.13 to the Boc-protected-β-amino acid ester VIII.14.

VIII.2.1.3 Hydrolysis of the methyl ester:

The hydrolysis of the methyl ester VIII.13 to the carboxylic acid proceeds without problems with the aid of NaOH (scheme VIII.4).

H Me H Me 4 eq NaOH, H O/MeOH (2/1), N N 2 Boc COOH Boc COOMe 50°C, 2 h.

96% VIII.14 VIII.16

Scheme VII.4 Hydrolysis of the methyl ester in VIII.14 to the corresponding carboxylic acid.

133

Part 2: Results and discussion

VIII.2.1.4 Boc-removal

Treatment of the Boc protected amino acid VIII.16 with acidic conditions (HCl), smoothly resulted in 99% yield of VIII.17, which could be used in the next step without further intermediate purification (scheme VIII.5).

H Me N Conc. HCl/dioxane 1/9, 7 h, r.t Me Boc COOH HCl H N . 2 COOH

99% VIII.16 VIII.17

Scheme VIII.5 Boc-deprotection of the carboxylic acid using a mixture of HCl/dioxane.

VIII.2.1.5 Fmoc-protection

Final Fmoc protection using Fmoc-OSu in presence of Na2CO3 only afforded the Fmoc-protected- β2,2-amino acid VIII.16 in 38% yield in addition to a mixture of compounds. An alternative procedure using Fmoc-Cl in presence of DIPEA and TMSCl delivered the protected product in better yield (44%; scheme VIII.6).

H Me i) 2 eq TMSiCl, 1 eq DIPEA, CH Cl , reflux 6 h. HCl.H2N 2 2 N COOH ii) 2 eq DIPEA, 1 eq FmocCl, 0 C-r.t, o.n Fmoc COOH 44% VIII.17 VIII.8

Scheme VIII.6: Fmoc protection of β2,2-amino acid VIII.17.

The rather low yield of N-protection could possibly be attributed to sterical effects.

134

Part 2: Results and discussion

VIII.3 SOLID PHASE SYNTHESIS OF A MODEL 3,3-DISUBSTITUTED-1,5- BENZODIAZOCINE-2,6-DIONES

In a first step, it was decided to test the synthesis route on a simple model compound.

H O N

N O VIII.26

Figure VIII.3 Model 3,3-disubstituted-1,5-benzodiazocine-2,6-dione VIII.26.

VIII.3.1 Coupling of Fmoc-β2,2-amino acid on Wang resin and Fmoc removal

Fmoc-β2,2-amino acid is coupled to Wang resin-using the same Steglich conditions (DIC/DMAP) of previous Fmoc-β2/β3-amino acid loading steps followed by a capping procedure. The efficiency of the coupling step was determined via quantitative UV-Fmoc determination after extensive drying of the beads. The loading was found to be 0.48 mmol.g-1 significantly lower compared to the usual loadings of around 0.65 mmol.g-1, indicating a steric effect of the quaternary α- substitution pattern on the β2,2-amino acid building block. Fmoc removal is achieved by treatment with 4-methylpiperidine in DMF to deliver the resin VIII.19, (scheme VIII.7).

135

Part 2: Results and discussion

1. a) 2 eq Fmoc-AA-OH VIII.8, 2 eq DIC, 20 min, CH2Cl2, 0°C O b) 0.2 eq DMAP, CH Cl , r.t, 24h. OH 2 2 O NHFmoc

2. Ac2O/DIPEA/CH2Cl2 (1/1/3), r.t, 2 x 1h OAc VIII.6

VIII.18

20% 4-methylpiperidine in DMF, 2 x 20 min, rt

O NH2 OAc

VIII.19

Scheme VIII.7 Coupling of Fmoc-β2,2-amino acid VIII.8 to Wang resin and Fmoc removal using a solution of 20% 4-methylpiperidine in DMF.

VIII.3.2 Mitsunobu-Fukuyama alkylation sequence

Again as before, we wished to introduce diversity at the N-5 position via a Mitsunobu- Fukuyama N-monoalkylation procedure. To this end, the resin-bound primary amine is treated with 2- nitrobenzenesulfonyl chloride (5 eq) in the presence of collidine (10 eq), followed by alkylation with MeOH (10 eq) in the presence of triphenylphosphine (5 eq, 0.2 M) and diisopropyl azodicarboxylate (5 eq, 0.2 M). To ensure complete alkylation, this step was repeated two more times. Nosyl-removal was achieved with the aid of β-mercaptoethanol and DBU to afford resin- bound model secondary amine VIII.22, as verified by LCMS analysis.

136

Part 2: Results and discussion

o-Ns

O O O O NO2 5 eq o-Ns-Cl, 10 eq collidine S O NH2 O N Me CH Cl , r.t, 2x1h 2 2 MeH

VIII.19 VIII.20

10 eq MeOH, 5 eq Ph3P, 5 eq DIAD, DCE, r.t, 3x2 h

O O O O NO2 OH S O NH 5 eq HS , 2.5 eq DBU O N MeMe MeMe DMF, r.t, 2 x 30 min

VIII.22 VIII.21

Scheme VII.8: Mitsnubo-Fuakayama reaction of VIII.19 with MeOH.

VIII.3.3 Coupling of Fmoc-anthranilic acid derivatives and Fmoc removal Coupling of the Fmoc-anthranilic acid to resin-bound amine VIII.22 was achieved using the symmetrical anhydride method formed from N-Fmoc-anthranilic acid (10 eq) and diisopropylcarbodiimide (DIC 5 eq). A full conversion (monitoring via LCMS) was observed after double treatment. Fmoc-removal was accomplished by treatment of VIII.23 with a solution of 20% 4-methylpiperidine in DMF 2x20 min, delivering the resin-bound dipeptide VIII.24. LCMS- analysis shows the clean formation of the expected compound.

137

Part 2: Results and discussion

O O O NHFmoc

O NH O N MeMe 10eq anthranilic acid, 5 eq DIC, MeMe CH2Cl2/DMF 9/1, r.t, o.n.

VIII.22 VIII.23

20 % 4-methylpiperidine in DMF 2x 20 min

O O NH2 O N MeMe

VIII.24

Scheme VIII.9 Coupling of anthranilic acid to resin-bound amine VIII.22 followed by Fmoc removal.

VIII.3.4 Attempts towards cyclization Previously at our laboratory, a successful cyclization/release step was observed using KOtBu in THF for compounds containing the β2,2-dimethyl substitution pattern. Application of these conditions to the resin bound amine VIII.24, however did not afford the desired cyclized product. No products could be detected in solution, neither at room temperature nor at 60 °C, and inspection of remaining resin-bound material by LCMS-analysis after TFA-cleavage, revealed mainly unreacted VIII.24 in the presence of a mixture of unknown impurities. Probably steric effects are responsible for this disappointing results. Alternatively the solution-phase cyclization was investigated next, in which the dipeptide VIII.25 was cleaved using TFA; purified via column chromatography (94% yield from VIII.18) and submitted to cyclization conditions in solution.

138

Part 2: Results and discussion

O O NH2 i) 2 eq ClCOOMe, 4 eq DIPEA, HO N TFA/H2O 0.01 M DCM, r.t, 1h 95/5 or 94% from VIII.18 ii) 3 eq DCC-PS, 0.01M DCM, r.t, 1h

X VIII.25 H O O O NH2 N KOt-Bu, THF O N r.t or 60 °C N O

VIII.24 VIII.26

Scheme VIII.10 Cyclization attempts to form 3,5-dimethyl-3-naphthylmethyl-1,5-benzodiazocine- 2,6-dione VIII.26.

Purified VIII.25 was treated initially on test scale with the earlier optimized conditions (ClCOOMe or polystyrene-bound DCC). The use of ClCOOMe in presence of DIPEA affords the cyclized VIII.26 in 29% relative purity, in addition to a mixture of compounds, while reaction with resin-bound DCC provided the product in 78% relative purity, along with 8% remaining starting material (figure VIII.4, peaks are integrated in LCMS at 214 nm).

Figure VIII.4 Crude LCMS sample of the cyclized product VIII.26 using resin-bound DCC.

Repeating the DCC-mediated reaction on bigger scale, delivered the expected benzodiazocinedione in 60 % yield.

139

Part 2: Results and discussion

VIII.4 CONCLUSION

Based on the proposed solid phase synthesis strategy for the 3,3-disubstituted-1,5-benzodiazocine- 2,6-diones VIII.1, essential building blocks consist of N-Fmoc-anthranilic acids and N-Fmoc-3,3- dialkyl-3-aminopropionic acids. A model building block of the latter was successfully prepared in moderate overall yield (synthetic route is depicted in scheme VIII.11).

a NC COOMe b NC COOMe c NC COOMe NC COOMe Me 88% 92% 78%

VIII.9 VIII.27 VIII.12 VIII.13

Boc COOMe Boc COOH COOH d N e N f HCl.H2N g H Me H Me Me 60% 96% 99% 44%

VIII.14 VIII.16 VIII.17 Fmoc COOH N H Me

VIII.8 16% overall yield, 7 steps

Reagents and conditions: a) 1 eq 2-naphthaldehyde, 0.1 eq AcOH, 0.04 eq piperidine, dioxane, r.t., o.n. b) 1.1 eq NaCNBH3, 0.4 M AcOH, MeOH/CH2Cl2 5/1, r.t, 1h. c) 5 eq MeI, 2 eq NaOMe, THF, 1M, 1h. d) 0.1 eq NiCl2.6H2O, 7 eq NaBH4, 2 eq Boc2O, MeOH, 0C to r.t., o.n. e) 4 eq NaOH, (H2O/MeOH 2/1), r.t to 50° C, 2h. f) Dioxane / concentrated HCl 9/1, r.t, 7h. g)i) 2 eq TMSiCl, 1 eq DIPEA, CH2Cl2, reflux 6 h. ii) 2 eq DIPEA, 1 eq FmocCl, 0 C-r.t, o.n. Scheme VIII.11 Synthetic route to β2,2-amino acid building block VIII.16.

Using this model β2,2-amino acid building block, the solid-phase synthesis of model benzodiazocinedione VIII.26 was investigated (detailed reaction conditions are depicted in scheme VIII.12).

140

Part 2: Results and discussion

O O O a b c o-Ns OH O NHFmoc O NH2 O N Me Me Me H

VIII.6 VIII.18 VIII.19 VIII.20 O O O O NHFmoc d o-Ns e f b O N O NH O N MeMe MeMe MeMe

VIII.21 VIII.22 VIII.23

O O O NH2 O O NH2 H N Me g h O N HO N MeMe 94% from VIII.18 MeMe 60% N O Me

VIII.24 VIII.25 VIII.26

Reagents and Conditions: a)1) 2eq VIII.8, 2 eq DIC, CH2Cl2, 0.2 eq DMAP, 24h. 2) Ac2O/ DIPEA/CH2Cl2 1/1/3 , 2 x 2 h. b) 20% 4-methylpiperidine in DMF, 2 x 20 min. c) 5eq o-NsCl, 10eq collidine, CH2Cl2, 2 x 1 h. d) 10 eq MeOH, 5 eq Ph3P, 5 eq DIAD, DCE, 3 x 2 h. e) 2.5 eq DBU, 5 eq 2-mercaptoethanol, DMF, 2 x 30 min. f) 10 eq Fmoc- anthranilic acid, 5 eq DIC, CH2Cl2/DMF 9/1, 24 h. g) TFA/H2O 95/5, 2 x 1 h. h) polystyrene-bound-DCC, CH2Cl2, 1h.

Scheme VIII.12 Solid-phase synthetic route towards VIII.26

Unfortunately, the anticipated direct cyclization/release approach from precursor VIII.24 using KOtBu did not yield the expected benzodiazocinedione. Alternatively, the solution-phase cyclization using resin-bound DCC delivered the target bislactam. In conclusion, it seems the increased steric congestion in the cyclization precursor hampers the general application of the KOtBu-mediated cyclization methodology as used before for 3,3-dimethylated-1,5- benzodiazocine-2,6-diones.

141

Part 2: Results and discussion

IX CONFORMATIONAL AND MODELING STUDY

IX.1 INTRODUCTION

Atropisomerism, a term coined by Kuhn in 19331, refers to stereoisomerism resulting from hindered rotation around a single bond such that the isolation of individual conformers is possible (in Greek the word atropos means without rotation). The optical activity due to axial chirality was first reported by Christie and Kenner in 19222, after a single enantiomer of 6,6'-dinitro-2,2'- diphenic acid IX.1 was isolated from the racemic mixture via diastereoselective crystallization with a chiral resolving agent.

HOOC NO HOOC NO2 2 HOOC NO2 HOOC NO2

IX.1

Figure IX.1 Atropisomerism of 6,6'-dinitro-2,2'-diphenic acid IX.1

Atropisomers are recognized as physically separable species3 when - at a given temperature - they have a half-life of < 1000 s. The dynamic interconversion between two isomers is influenced by sterical hinderance, electronic influences, temperature and solvent. To describe the atropisomers according to their level of interconversion at a certain temperature, half-lives were introduced (figure IX.2). Based on correlation of calculated energy barriers and rotational rates, atropisomers are categorized into three classes; class 1: compounds show no axial chirality, do not have atropisomers with ΔErot > ~ 20 kcal/mol, class 2: shows a noticeable interconversion over time

ΔErot ~ 20-30 kcal/mol and may be subdivided into two subclasses, rapid interconversion / delayed interconversion, class 3: compounds show stability overtime, have a rotational energy barrier ΔErot > ~ 30 kcal/mol and can be isolated as (enantiomerically) pure compounds.

142

Part 2: Results and discussion

r r Class 3: single compounds a

e Class 2: mixture

i Class 1: single compounds

o T

Figure IX.2 Classification of atropisomers based on torsion rotation energy barrier.

This phenomenon can have a major impact on the properties of the compounds, for example in pharmaceutical applications4. In this respect, the ring inversion of diazepam and analogues has been investigated by Gilman and co-workers5. They found that the interconversion energy barrier increases by increasing the substitution on N1 (figure IX.3). Diazepam (IX.2, R=Me), has an interconversion barrier of 73.6 kJ/mol and can thus considered to be a slow interconverting mixture of both conformers. However, when a tert-butyl group is introduced at N1, the interconversion energy barrier is higher than 100.4 kJ/mol, and the mixture can be separated into stable isomers.

R R O O N N

Cl N Cl N

IX.2 IX.3

Figure IX.3 Inversion of N-substituteddiazepam

Table IX.1 Ring inversion energy barriers

R ΔErot[kJ/mol] H 50.2 Me 73.6 iPr 88.3 tBu <100.4

143

Part 2: Results and discussion

IX.2 CONFORMATIONAL BEHAVIOUR OF EIGHT-MEMBERED BENZO- (BIS)LACTAM COMPOUNDS – RELEVANT LITERATURE

The major conformation influencing moieties in the 1,5-benzodiazocine-2,6-dione skeleton 2 comprise the sp -hybridized diazocine-atoms; being the two planar amides (N1-C2 and N5-C6) along the C-C double bond at the annelation position, creating two possible cases of axial chirality. The conformational behavior of related eight-membered benzo-(bis)lactam compounds have already been studied in literature, so looking into these studies of reported systems might give an insight in the 3D structure of our scaffold.

Because of the structural similarity in the literature the comparison of lactams is often made with the analogous carbocyclic parent systems (alkenes or benzo-annulated). To illustrate the influence of one conjugated benzene-amide moiety, it is interesting to discuss the conformation of benzolactams IX.4 and IX.5. An extensive conformational NMR/DFT study was recently performed by Witosinska6 for these structures (IX.4, IX.5, figure IX.4). It was shown that the amide bonds are planar and adapt the E configuration (cis), leading to a major conformer (98%) displaying a ground state twist-boat-chair conformation similar to the conformation of (Z,Z)-1,3- cyclooctadiene IX.36. Because of the atropoisomerism phenomena due to the presence of the sp2- sp2 chiral axis of the benzamide bond, the molecule can interconvert to its “flipped” mirror conformation, for which energy barriers of 59-66 kJmol-1 have been determined by DNMR. Presence of an N-methyl substituent increases the energy barriers for conformational processes; in fact, at room temperature, lactam IX.4b is conformationally locked and exists as a racemic mixture of uninterchangeable chiral conformers.

As an illustration, the X-ray structure of IX.4a is shown in figure IX.4 (left), along the DFT- predicted conformation (right). To have a visual insight on the possible conformational processes, the proposed mechanisms are shown for (Z,Z)-1,3-cyclooctadiene (figure IX.5).

144

Part 2: Results and discussion

R O O R N N

IX.4a IX.4b IX.5a IX.5b

R: H Me H Me

G : ~59 ~100 ~66 ~88

Figure IX.4 Top: Structure of the compounds IX.4, IX.5 and their experimentally determined barriers for dynamic processes from DNMR. Bottom: Structure of IX.4a as determined by x-ray crystallographic analysis (bottom left) and computed (DFT) lowest energy conformation (bottom right)6.

3 4 2 5 6 1 8 7 IX.3

(Z,Z)-1,3-cyclooctadiene

Figure IX.5 (Z,Z)-1,3-Cyclooctadiene as a carbocyclic conformational model7.

145

Part 2: Results and discussion

In contrast to the twist-boat chair conformations of these benzoannelated monolactam benzodiazocines, the introduction of an extra Л-bond (and thus extra axis of chirality) generally gives rise to compounds in the boat or twist-boat conformation. In principle, according to IUPAC- rule, these 8-membered ring conformations should be addressed as “tub” conformation, but in this dissertation we follow the commonly used “boat” terminology.

Again, the carbacyclic parent system may serve as a starting point to describe the expected conformational possibilities. It has to be kept in mind of course that accompanying energy values will be highly depending on the individual compounds and could thus dramatically shift equilibria between conformational states.

D D

9 11 IX.79,10 IX.8 IX.9 9 enantiomers could be separated by chromatography Tcoal= -136C Tcoal= -98C 10  and consequently racemized at 49-64 C, giving Tcoal= -145C G kJ/mole  GkJ/mole9 =26.0 Hz G kJ/mole GkJ/mole10 Figure IX.6 Structures of IX.7, IX.8, IX.9. Experimentally measured barriers (100 MHz,

CHF2Cl/CH2CHCl 1/1 solution for IX.7 and IX.8.

5 6

4 7 3 8 2 1

IX.10

Figure IX.7 1,3,5-cyclooctatriene as a conformational model.

As can be seen from figure IX.7, twist-boat (C7-C8 staggered) conformations are considered to be the ground states, which are thought to interconvert through pseudo-rotation via a boat (C7-C8

146

Part 2: Results and discussion eclipsed) transition state or through conformational ring inversion via high energetic half chair transition states8.

Experimental values9,10 for these energy states and barriers have been obtained for a number of related systems, as summarized in figure IX.6 and figure IX.8.

Comparing IX.7 and IX.8, the 10 kJ/mol higher barrier can be attributed to an increase in the angle strain in the transition state for inversion, due to the fusion of the benzene ring. During the transition state the three double bonds approach planarity, causing an increase of internal bond angles; fusion of a benzene ring will render this expansion more difficult, therefore increasing the barrier.

From NMR analysis on a small series of pyrazolobenzodiazocines IX.11, Gyomore et. al.12,13 concluded a boat-like conformational ground state and obtained x-ray crystallography data on

IX.12 (R3=Et), showing the twisted-boat geometry (figure IX.9).

O O O O NH Me NH NH R1 NH Me Me R2 Me N N N N N N R3 R3 O MeO OMe OR4 OH O

R3= H, Tcoal=368 K Tcoal=355 K x-ray  R3= Me, Tcoal=376 K G kJ/mole twisted-boat R3= Et, Tcoal>390 K; no broadening of peaks

IX.11 IX.12 IX.13 IX.14 Figure IX.8 Examples of studied related 8-membered ring systems.

Here, the nature of the R3 substituents seems to be important for the level of conformational freedom. Where H- or Me-substituted compounds (IX.12) showed ring-interconversion (Tcoal= 268 K, 376K, respectively), ethyl-substituted IX.12 was completely conformationally locked as no 1H NMR line broadening could be observed at temperature up to 390 K. Olivia-Madriol observed a twist-boat conformation for compound IX.14 through X-ray analysis14.

147

Part 2: Results and discussion

Figure IX.9 Structure of IX.12, as determined by x-ray crystallographic analysis.

The boat-shaped conformation was also devised for the eight-membered homologue of diazepam (IX.15), a well-known benzodiazepine drug. The x-ray structure is shown in figure IX.10.

Me O N

Cl N

IX.15

Figure IX.10 Structure of IX.15 as determined by x-ray crystallographic analysis.

148

Part 2: Results and discussion

Elguero and colleagues15 studied the conformation of the, for us, strongly similar 3,4-dihydro-1,6- benzodiazocine-2,5-dions nucleus. It was demonstrated from 1H NMR that the ground states involved are true boat conformations (thus C3-C4 eclipsed), confirmed by molecular modeling performed at our laboratory (Jan Goeman). The boat-boat interconversion process was shown to be influenced by the substitution pattern on the amide functionalities. For N-unsubstituted derivatives IX.16, a rapid interconversion was observed at room temperature, whereas mono- methylated IX.17 showed broadening of the 1H NMR signals above 120°C, (unfortunately a full analysis was not possible as the compound decomposed at 140 °C).

H O Me O Me O N N R N R

N N N H H O O Me O IX.16 IX.17 R=H IX.18 R=Me IX.19

R=H, Me broadening of peaks R=H one conformer (boat) rapid boat-boat above 120 °C 1H NMR broadening of peaks at 180 C interconversion at decomposition at 140 °C  ambient temp. (G kJ/mol) R=Me two conformers B1(endo)/B2(exo) Broadening of peaks at 180 C  G between100-105 kJ/mol

Figure IX.11 Structures of compounds IX.16, IX.17, IX.18 and IX.19.

For N,N`-dimethyl derivatives IX.18 and IX.19, broadening of the 1H NMR peaks only occurred at 180 °C, resulting in free energy of activation estimates over 100 kJ/mol. For trimethyl compound

IX.19 two conformers B1 and B2 are postulated, in which the C3 methyl group is located in a pseudo-axial (“endo”) or pseudo-equatorial (“exo”) position, respectively. The B1/B2 ratio after N- methylation of IX.16 (R=Me) was found to be 7/3, which equilibrated over time and temperature to the thermodynamic equilibrium ratio of 2/8. Own modeling experiments confirm these structures.

149

Part 2: Results and discussion

Figure IX.12 Top: Reported boat conformations of IX.19 by Elguero. Bottom: Results of our modelling experiment of IX.19: Left: ground state (in agreement with conformation B2); right: next conformer (calculated +2.17 kJ/mol), in agreement with conformer B1. From this energy difference, a 71/29 ratio can be expected at thermodynamic equilibrium at 298K.

IX.3 CONFORMATIONAL ANALYSIS OF 1,5-BENZODIAZOCINE-2,6-DIONES

IX.3.1 Modeling of 3-substituted-1,5-benzodiazocine-2,6-diones

Four compounds were subjected to a modeling experiment (in CHCl3) using Maestro software and gave overall general conformational results. As illustrated in figure IX.13 for IX.20, the energetic ground state adopts a clear twist-boat conformation in which the C3-C4 bond is clearly present in the staggered rotamer (figure IX.13, left), differing from the above-discussed isomeric 1,6- benzodiazocine-2,5-diones which seem to adopt a true boat conformation (C3-C4: eclipsed).

Noteworthy is the presence of the C3-methyl group in the pseudo-axial position (“endo”). The next calculated energetic minimum (4.659 kJ mol-1) is an alternative twist-boat conformation (figure

IX.13, right), in which the C3-methyl group assumes the pseudo-equatorial orientation (“exo”). These clearly arise from a ring inversion, for which a relative ratio of 87/13 can be calculated (from ΔG°=-RTlinK). Other calculated configurations had relative energies higher than 10 kJ.mol-1 and

150

Part 2: Results and discussion were therefore not considered. The other compounds IX.21, IX.22, IX.23 show the same conformational behavior (see table IX.1). From the obtained energetic values, it seems that for larger substituents on C3 and N5, the calculated energy difference would lead to almost fully populated ground state conformations at thermodynamic equilibrium. However, being at thermodynamic equilibrium requires equal macroscopic rates of dynamic interconversion between the different conformations, which is only possible if the energy barriers for interconversion can be overcome at the given temperature. If barriers are too high, conformers can become conformationally locked and can lead to stable isomers which can eventually be isolated. Such behavior can have profound effects on the NMR spectra at hand, as coalescence effects will appear when conformational dynamics are approaching the NMR time scale. Actually, from these line- broadening effects in 1H NMR, experimental interconversion barriers can be deduced for these conformational processes.

O HN

N O

IX.20

Figure IX.13 Two lowest energy conformers for compound IX.20. A. lowest energy conformer; B. conformer with relative energy: + 4.659 kJ.mol-1.

151

Part 2: Results and discussion

Table IX.1 Results of modeled 3-substituted-1,5-benzodiazocine-2,6-diones IX.20-IX.23.

Compound ΔG° Ratio at Substitution on Substitution on 298 K C3 (Ground C3 (Inverted state) boat) O 4.659 kJ.mol-1 87/13 pseudo-equatorial pseudo-axial HN IX.20 N O O 8.807 kJ.mol-1 97/3 pseudo-equatorial pseudo-axial HN

N O IX.21 O 9.172 kJ.mol-1 98/2 pseudo-equatorial pseudo-axial HN IX.22 N O

O 9.68 kJ.mol-1 98/2 pseudo-equatorial pseudo-axial HN IX.23 N O

IX.3.2 Modeling of 4-substituted-1,5-benzodiazocine-2,6-diones

Three 4-substituted-1,5-benzodiazocine-2,6-diones were also submitted to molecular modeling (in

CHCl3). Again, results were consistent over this series. Visualized for analogue IX.24, a twist- boat conformation is the energetically most stable structure (figure IX.14, A; 0,0 kJ.mol-1). However, in contrast to the above 3-substituted derivatives, the methyl substituent at C4 is now occupying a pseudo-equatorial orientation (“exo”). The next energy minimum corresponds to the ring-inverted conformer, in which the C4-methyl group is oriented pseudo-axially (”endo”, figure IX.14, B; 2.605 kJ.mol-1). The other compounds IX.25 and IX.26 show the same behavior (see table IX.2).

152

Part 2: Results and discussion

O HN

N O

IX.24

Figure IX.14 Two lowest energy conformers for compound IX.24. A. lowest energy conformer; B. conformer with relative energy: + 2.605 kJ.mol-1.

Table IX.2 Results of modeled 3-substituted-1,5-benzodiazocine-2,6-diones IX.24-IX.26.

Compound ΔG° Ratio Substitution on C4 Substitution on C4 at 298 (Ground state) (Inverted boat) K O 2.605 kJ.mol-1 74/26 pseudo-axial pseudo-equatorial HN

N O IX.24

O 1.761 kJ.mol-1 67/33 pseudo-axial pseudo-equatorial HN

N IX.25 O

153

Part 2: Results and discussion

O 5.170 kJ.mol-1 89/11 pseudo-axial pseudo-equatorial HN

N IX.26 O

IX.3.3 Experimental data on the synthesized 1,5-benzodiazocine-2,6-diones

As we have been using racemic beta amino acids for our libraries, the final 1,5-benzodiazocine- 2,6-dione derivatives are isolated as racemates. Because of the conformational behavior discussed above, in case of slow ring interconversions, we can expect a mixture of possible isomers at ambient temperature in NMR experiments. As these are present as two diastereomeric pairs of enantiomers, we expect two main diastereoisomeric boat-type conformations. Depending on the energy barriers, higher temperatures could then lead to coalescence or even further simplification as the average of the conformational equilibrium will be visible. If the barriers of interconversion would be sufficiently high, the isolation of the isomers at room temperature would even become achievable. In figure IX.15 an overview on the expected conformers, along their enantiomeric/diastereomeric relationships. For the assignment of the (M)/(P)-helical descriptors, we base ourselves on existing nomenclature for the well-known 1,4-benzodiazepines, such as the closely related 1,4-benzodiazepine-2,5-diones 16. Assignment is thus relying on the positive (P = Plus, clockwise rotation) or negative (M = Minus, counterclockwise rotation) dihedral angle along the atom chain C2-N1-C10a-C6a.

154

Part 2: Results and discussion

H H R H (S) (R) 6a diastereoisomers H R H H 10a N N N N 1 2 O R O R H O H O (P)-conformer (P)-conformer

diastereoisomers enantiomers enantiomers diastereoisomers

O H O H O R O R N N N N H H diastereoisomers R H (R) (S) H R H H

(M)-conformer (M)-conformer

Figure IX.15 Four possible conformers of 3-substituted-1,5-benzodiazodine-2,6-diones and their enantiomeric/diasteriomeric relationships, with assignment of (M)/(P)-helical descriptors.

The presence of multiple conformational states in the synthesized 1,5-benzodiazocine-2,6-diones becomes immediately clear from the corresponding complex 1H NMR and 13C NMR spectra. For more simple-substituted benzodiazocinediones, it is generally possible to visualize two main conformers in differing ratios. For analogues containing larger (and thus more rotation-prone) substituents, additional (albeit lowly abundant, < 5%) conformers can be detected/suspected. For ease of discussion, these minor conformers are omitted in the following paragraphs. To avoid issues of impurities, compounds were intensively purified (column chromatography, followed by reversed phase HPLC purification). Purities were generally over 95% (LCMS analysis at 214 nm). In the following section, a preliminary discussion is offered. A more detailed NMR study, along theoretical support, on the conformational behavior could be an interesting subject of future research.

In figure IX.16 an overview is given of the observed ratios of boat-type conformers, as determined by integration of the corresponding 1H NMR spectra.

155

Part 2: Results and discussion

O O O O O H O H H H H H N N N N N N

N N MeO N N N N O F O O O O O IX.21 IX.23 IX.27 O IX.22 IX.28 IX.29 Cl O 80/20 80/20 70/30 Cl ~75/25 ~80/20 82/18 OMe O O O H H H O N N N O H H N N Br N MeO N Me N N O O O MeO N O IX.31 IX.32 O IX.30 IX.33 IX.34 ~80/20 70/30 ~75/25 Cl F 70/30 could not be Cl determined*

O O H O H H O O H O N H H N N N N N

MeO MeO Br N N N Br N N N O O O O O O H O O IX.39 N IX.35 N IX.36 IX.24 IX.37 IX.38 ~60/40 ~60/40 ~60/40 ~67/33 could not be O 70/30 CF O determined* 3 O O H O H O H O H N H N N N N F3C

Br N Br N N N N O O O O O F IX.40 IX.41 IX.42 O IX.43 IX.44 H 80/20 OMe 87/13 50/50 80/20 H O 84/16 N N

N N O O IX.26 IX.45 75/25 ~65/35 Figure IX.16 Observed ratios of conformers for synthesized 1,5-benzodiazocine-2,6-diones, as determined from 1H NMR spectra. *could not be determined because of overlapping.

156

Part 2: Results and Discussion

IX.3.3.1 Analysis of 3-substituted-1,5-benzodiazocine-2,6-diones

As an example of a “simple” 3-substituted benzodiazocine, the 1H NMR spectrum of IX.23 is shown in figure IX.17. Two major conformers seem to be present at room temperature, in a ratio of around 80/20. The observed relatively broad peaks of the isobutyl group in the minor conformer (0.70-0.83 ppm, as compared to the sharp peaks in the major conformer, ~0.88 ppm) are probably due to the less restricted rotation of this side chain, as would be expected of an isobutyl group in the exo-position (see figure IX.13, conformer B (exo) versus A (endo)).

O HN

N O IX.23

2 CH3 isopropyl

NH CH3

Figure IX.17 Details of the 1H NMR spectrum of IX.23, recorded at room temperature in acetone- d6 (400 MHz), indicating the ratio of two conformers 8/2.

The observed 8/2 ratio is rather different compared to the predicted 98/2 ratio, based on the calculated energy difference between expected conformers (9.68 kJ/mol, see table IX.1). This could mean that either there is an error on the calculated value (although solvent effects can influence these values), or that the compound is not in thermodynamic equilibrium because of

156

Part 2: Results and Discussion large conformational interconversion barriers. In an attempt to verify the latter, a heating experiment was conducted under microwave conditions. Heating for 30 minutes at 150 °C and 5 minutes in 210 °C in DMSO, however, did not reveal any changes in the recorded NMR spectra. Although higher temperatures unfortunately led to decomposition, these results suggest the compound to be in thermodynamic equilibrium, when taking into account literature precedents on related ring systems. As such the difference in free energy between the conformers may be estimated to be around 3.4 kJ mol-1. The barrier for interconversion could be determined in the future using DNMR.

As an example of a “more complicated” derivative, the 1H NMR spectrum of compound IX.27 shows multiple conformations (figure IX.18). Because of the larger N-substituent, it is also predicted to be more conformationally restricted because of higher energy barriers. Indeed, it is actually the only compound from the synthesized 3-substituted benzodiazocines for which the diastereomeric conformers are found to be resolved in the LCMS analysis. Unfortunately, attempts to separate the isomers by column chromatography or HPLC were not successful. Again, as the observed ~7/3 - 8/2 ratio of the major isomers is rather different from the predicted values of related structures (~98/2, table IX.1), a heating experiment under microwave conditions was conducted. To this end, the sample was heated consecutively at 150 °C, 170 °C, 190°C and 210°C for five minutes each, taking samples between each experiment for LCMS-analysis (integration at 254 nm). As depicted in figure IX.20, it was found that the ratio (82/18) remained unchanged throughout the experiments, indicating a very high energy barrier for ring-interconversion. Unfortunately, further heating at 230°C caused complete decomposition of the compound. Alternatively, for this product a 1H NMR experiment was performed at higher temperature (130 °C in DMSO, figure IX.19) in an attempt to visualize coalescence phenomena; however, only marginal broadening of the peaks could be observed which again point to considerable ring- interconversion barriers. Because of technical reasons, recordings at higher temperatures could not be performed.

157

Part 2: Results and Discussion

H O N

N F O O IX.27 O

DMSO-d6 at room temperature

1 Figure IX.18: Detailed HNMR spectra of IX.27 in DMSO-d6 at room temperature.

DMSO-d6 at 130 °C

1 Figure IX.19: Detailed HNMR spectra of IX.27 in DMSO-d6 130 °C.

158

Part 2: Results and Discussion

Room temperature, MeOH, 82/18

170 °C, mw, 5 min, DMSO, 83/17

190 °C, mw, 5 min, DMSO, 83/17

210 °C, mw, 5 min, DMSO, 83/17

230 °C, mw, 5 min, DMSO, decomposed

Figure IX.20 Integration of two conformer peaks in LCMS at 254 nm at room temperature and after heating at 170 °C, 190°C, 210 °C and 230 °C for compound IX.27.

159

Part 2: Results and Discussion

IX.3.3.2 Analysis of 4-substituted-1,5-benzodiazocine-2,6-diones

The main conformers of 4-substituted-1,5-benzodiazocine-2,6-diones were integrated in the 1H NMR spectra showing conformer ratios between 50/50 and 87/13 (figure IX.16). A detailed NMR analysis for the simple derivative IX.24 is illustrated in figure IX.21.

H O N

N O IX.24

1 Figure IX.21 HNMR spectrum of IX.24 in acetone-d6 at room temperature.

For this compound, two conformers are observed in approximately 6/4 ratio. After a detailed analysis of observed coupling constants, we assign the (P)-helical descriptor to the major conformer, while the minor conformer involves the inverted (M)-structure. The corresponding Newman projections are depicted in figure IX.22 (for the R-enantiomer). These results are consistent with the predicted conformations, shown in figure IX.14.

160

Part 2: Results and Discussion

Major Minor

O O H 2.63 (dd, J=16.5/12.1Hz) H 2.25 (dd, J=12.5/11.3Hz) N H N H H 2.83 (br.dd, J=16.3/5.8 Hz) H 2.40 (dd, J=12.5/8.5 Hz) R H 4.09 (ddq,J=6.4/(3x)6.4/12.4Hz) R H 3.86 (ddq,J=11.2/8.5/(x3)6.6 Hz) N Me N Me 1.20 (d, J=6.7Hz) 1.15 (d, J=6.6Hz) O Me O Me 2.96 3.06

O Me O N HN Me H H H H Me HN H H N O O Me (P)-(R)-IX.24 (M)-(R)-IX.24

1.15 O Me 1.20 N 2.63 2.25 Me H H H 2.40 2.63 Me 1.20 N H O N H3.86 ~ 90 J~0, H H H not observed O H 4.09 2.83 2.83 4.09

Karplus Equation

H H

H J

0 90 Φ[degree] Figure IX.22 Newman projection of the major and minor conformers of compound IX.24.

Again, the observed ratio (~6/4) is somewhat differing from the calculated 74/26 (table IX.2), so we wished to verify if the observed conformers are actually in thermodynamic equilibrium.

Thus, again a heating experiment under microwave irradiation was conducted. A room temperature 1H NMR spectrum was recorded after heating at 150 °C and 210 °C in DMSO, but no change in

161

Part 2: Results and Discussion the conformation ratio was observed. Unfortunately the compound decomposed at higher temperature.

Next, an NMR study was performed by recording 1H NMR spectra at various temperatures (from 298 K till 398 K, figure IX.23). From the observed coalescence phenomena, the activation energy can be estimated17 using the following equation;

≠ 푇푐 ΔG = aTc [9.972 + log ] ∆휈

Tc: The coalescence temperature

a= 1.914 x 10-2 for units kJ/mol

Δν: difference in chemical shift (in Hz) between distinguished signals

1 Figure IX.23 HNMR spectra at different temperatures for compound IX.24 (DMSO-d6-500

MHz). Left: N-H signals; Middel: N5-CH3 signals (the migrating large singlet is attributable to water in the sample).

Potential errors on the estimation of this values arise from the (sometimes difficult) determination of Tc and Δν. Often the isotropic chemical shifts of the exchanging species are temperature dependent, so Δν changes with temperature and error in ΔG≠ can become large. Examination of the 1H NMR experiment at different temperatures revealed that the coalescence temperature ranges

162

Part 2: Results and Discussion from 363 K to 388 K, depending on the signals of interest (see figure IX.23). Calculation of activation energy at coalescence for the different peaks through the above equation gives an average value of ΔG≠ = 77.6 kJ/mol, indicating a rapid interconversion between the conformers at room temperature and verifying that they are in thermodynamic equilibrium.

IX.4 CONCLUSION

A conformational analysis of representative members for the two synthesized libraries (3- substituted and 4-substituted 1,5-benzodiazocine-2,6-dione) was performed via a computational modeling experiment which clearly show two ring-inverted twist-boat conformations (differing from the literature-discussed isomeric 1,6-benzodiazocine-2,5-diones which adopt true boat conformations). For the 3-substituted 1,5-benzodiazocine-2,6-dione; the C3-substituent is present in the pseudo axial position (“endo”) in the ground state, while for the 4-substituted 1,5- benzodiazocine-2,6-dione the C4-substituent adopts a pseudo equatorial orientation (“exo”). The “endo” and “exo” conformers form a pair of “ring-flipped” conformational states with calculated energy differences of 1-10 kJ.mol-1 depending on the specific structure. The presence of these conformational states in the synthesized 1,5-benzodiazocine-2,6-diones was clear from the corresponding complex 1H NMR and 13C NMR spectra showing ratios varying between ~ 50/50 to 90/10.

From the observed coalescence phenomena in a series of 1H NMR experiments at temperatures from 298 K to 398 K of 4,5-dimethyl-1,5-benzodiazocine-2,6-dione IX.24, a conformational energy barrier of 77.6 kJ.mol-1 was estimated.

However, more complexly substituted library members only display marginal line broadening effects in 1H NMR spectra at high temperature (130 °C), therefore revealing a significantly higher rotational barrier. For these compounds, 1H NMR spectra were recorded after heating the samples up to 210 °C. However, no changes in conformer ratios were observed. In this case, peak ratios were also found to be unchanged after heating the sample to temperatures up to 210 °C. As a result, thermodynamic equilibrium between the two boat conformers can be suggested. Interestingly, in one compound IX.27 diastereomeric conformers were visible in LCMS as two peaks which

163

Part 2: Results and Discussion suggest that this compound exists as two stable atropisomers. Preparative separation, however, was not successful.

164

Part 2: Results and Discussion

References

1 Kuhn, R. Stereochemie (Ed.: K. Freudenberg), 1933, 803

2 Christie, C. H.; Kenner, J. Chem. Soc. 1922, 121, 614

3 Oki, M. Top. Stereochem. 1983, 17, 1.

4 Testa, B.; Vistoli, E.; Pedretti, A.; Eur. J. Pharm. Sci 2016, 88, 101-123.

5 Gilman, N. W.; Rosen P.; Earley, J. V.; Cook, C.; Todaro, L. J. Am. Chem. Soc. 1990, 112, 3969-3979.

6 Witosinska, A.; Musielak, B.; Serda, P.; Owinska, M.; Rys, B. J. Org. Chem. 2012, 77, 9784- 9794.

7 Yavari, I.; Kabiri-Fard, H.; Moradi, S. J. Molec. Str. (Theochem) 2003, 623, 237-244.

8 Anet F.; Yavari, I. Tetrahedron Lett. 1975, 4221-4224.

9 Buchanan, G. W. Tetrahedron Lett. 1972, 8, 665-668.

10 St-Jacques, M.; Prudꞌhomme, R. Tetrahedron Lett. 1970, 55, 4833-4836.

11 Rashidi-Ranjbar, P.; Sandstrom, J. Tetrahedron Lett. 1987, 28(14), 1537-1540.

12 Gyomore, A.; Kovacs, Z.; Nagy, T.; Kudar, V.; Szabo, S.; Csampai, A. Tetrahedron 2008, 64, 10837-10848.

13 Gyomore, A.; Csampai, A.; Holzbauer, T.; Czugler, M. Tetrahedron 2011, 67, 2979-2990.

14 Oliva-Madrid, M.; García-Lopez, J.; Saura-Llamas, I.; Bautista, D. Organometallics 2014, 33, 6420−6430.

15 Elgueroa, J.; Fruchier, A.; Llouquet, G.; and Marzin, C. Can. J. Chem. 1976, 54, 1135.

16 Kremen, F.; Gazvoda, M.; Kafka, S.; Proisl, K.; Srholcova, A.; Klasek, A.; Urankar, D.; Kosmrlj, J. J. Org. Chem. 2017, 82, 715−722.

17 http://chemnmr.colorado.edu/manuals/DNMR_Calculations.pdf

165

Part 2: Results and Discussion

X. GENERAL CONCLUSION AND FUTURE PERSPECTIVES

The overall goal of this work was to synthesize parallel combinatorial libraries of compounds based on the potentially interesting 1,5-benzodiazocine-2,6-dione scaffold, namely 3-substituted- 1,5-benzodiazocine-2,6-diones X.1, 4-substituted-1,5-benzodiazocine-2,6-diones X.2 and 3,3- disubstituted-1,5-benzodiazocine-2,6-diones X.3 (figure X.1) using commercially available or easily accessible building blocks, allowing a fast and efficient diversification of the target molecules.

O H H O H O N N N R1 R1 R R3 R3 R4 2 N N R1 N O R2 O R2 O R3 X.1 X.2 X.3

Figure X.1 Target compounds.

Our choice of methodology fell on the development of a solid-phase synthesis route. Although a solid-phase approach has some inherent disadvantages (such as a long initial synthesis development time and scale up problems), this strategy is still very useful in early stage drug discovery and development phases, as an available general synthesis would allow the fast generation of a large diversity of analogues around the skeleton of interest. As we choose the benzodiazocine framework with privileged-structure-like properties, we expect that the developed synthesis route can be applied in a broad range of different drug discovery programs targeting a large diversity of therapeutic biochemical targets.

The development and optimization time of the solid-phase synthesis is relatively slow as it involves careful consideration of the polymeric support and linker choice in order to be compatible with the synthesis strategy. Moreover, reaction monitoring is more time consuming as classical solution techniques are not possible to apply on solid-phase chemistry. Instead intermediates are typically destructively cleaved off from the resin to submit for conventional analytical techniques. However, after settlement of the carefully optimized synthetic multistep strategy, a fast access to a multitude of libraries is possible as intermediate purifications are limited to simple filtration and washing steps, which are amenable for automation.

166

Part 2: Results and Discussion

In an answer to our initial goal (goal 1, section IV), we have thoroughly investigated the synthesis of a model compound X.4, which was achieved through the solid-phase synthesis of an open-chain precursor, which was cyclized in solution phase using solid-supported DCC in good yield.

H O N

N O Bn X.4

Figure X.2 Model compound 5-benzyl-3,4-dihydro-1,5-benzodiazocine-2(1H),6(5H)-dione.

Using the developed synthetic procedure for the model compound, the synthetic route towards 3- substituted-1,5-benzodiazocine-2,6-diones was explored. Essential building blocks in this synthesis consist of N-Fmoc-anthranilic acid and N-Fmoc-β2-amino acid derivatives and commercially available alcohols. A synthetic route towards the β2-amino acid building blocks was developed starting from commercially available methyl cyanoacetate and appropriate aldehyde. In this way three N-Fmoc-β2-amino acids building blocks were synthesized, and applied in a library of 13 3-substituted-1,5-benzodiazocine-2,6-dione diversified on three positions with moderate to good overall yield for solid phase synthesis and low to moderate cyclization yields.

Our next goal consisted of 4-substituted-1,5-benzodiazocine-2,6-diones X.2. A set of three commercially available β3-amino acids was used as building blocks in an essentially similar route. However, a double coupling procedure of N-Fmoc anthranilic acid was found to be necessary due to steric reasons. In this way, a library of 11 4-substituted-1,5-benzodiazocine-2,6-dione diversified on 3 positions was synthesized with moderate to good overall yield for solid phase synthesis and low to moderate cyclization yields comparable to the 3-substituted-1,5- benzodiazocine-2,6-diones.

The last challenging compound that was tackled consisted of the 3,3-disubstituted-1,5- benzodiazocine-2,6-dione. An essential model β2,2-amino acid building block was synthesized with difficulty starting from methyl cyanoacetate and appropriate aldehyde. Unfortunately the anticipated direct cyclization/release approach using KOtBu did not yield the expected benzodiazocinedione. Alternatively, the solution-phase cyclization using resin-bound DCC delivered the target bislactam in moderate yield. The direct cyclization/release methodology using

167

Part 2: Results and Discussion

KOtBu seems to be only applicable to more simple quaternary β2,2-amino acid building blocks, as demonstrated before in our laboratory for 3,3-dimethyl-1,5-benzodiazocine-2,6-diones.

Although the synthesis is expected to be applied to the synthesis of homochiral libraries, all compounds in this study were prepared from racemic β2-, β3- and β2,2-amino acids, delivering the corresponding racemic benzodiazocinediones. This is justifiable as this is an initial library synthesis demonstration and the racemic compounds can be screened as such in high-throughput screening platforms.

For the subsequent and more specific drug discovery programs, the synthesis of optically pure compounds will be desirable. This would require the use of optically active β-amino acids, which are only very limitedly commercially available, but for which synthetic strategies are becoming valuable in literature. As such β3-amino acids could be obtained from the corresponding α-amino acids, and β2-amino acids can be accessed from linear or cyclic β-alanine derivatives that can be alkylated.

Computational modeling experiments show two ring-inverted twist-boat conformations for representatives of both of the synthesized two libraries (3-substituted and 4-substituted-1,5- benzodiazocine-2,6-diones). The presence of these two conformational states was clear from the corresponding complex 1H NMR and 13C NMR spectra. Because of the importance of atropisomerism in pharmaceutical applications, a further study was conducted. For simple compound X.6 a series of 1H NMR experiments at different temperatures show coalescence effects from which an estimated interconversion energy barrier of 77.6 kJ.mol-1 was obtained. For more complex structures only marginal broadening of peaks in the 1H NMR spectra could be obtained up to 130 °C, indicative for significantly higher barriers between conformers; heating experiments up to 210 °C did not reveal any change in the observed ratio of conformers. Interestingly, both conformers of compound X.5 (as the only example from the synthesized libraries) were observed as separate peaks during LCMS analyses, suggesting that this structure consists of two co-existing stable atropoisomers.

168

Part 2: Results and Discussion

H O H O N N

N N F O O O O X.5 X.6

Figure X.3 Selected compounds showing high and low energy barriers as determined by 1H NMR heating experiments and LCMS analyses.

Further research is clearly necessary to gain more detailed insight in the conformational behavior of these potentially pharmaceutically interesting structures. In this regard, further investigation of N1-alkylated analogues and influences of cis/trans-C3,C4-disubstitution patterns could be useful.

169

Part 2: Results and Discussion

XI ENGLISH SUMMARY

XI.1 INTRODUCTION

A perceptive analysis of modern drug research highlights the importance of so called “privileged structures”. Before, new active compounds were typically sought in natural resources (plants, animals, microorganisms) or were obtained after random screening of individually synthesized “synthetic” compounds. The privileged structures term, was introduced to represent substructures that confer activity to two or more different receptors1. Molecules based upon these (sub)structures have been shown to yield a higher success rate in the discovery of new drug candidates that comply to Lipiniski’s rule2. An important class of privileged structures consist of a family of compounds built around the popular 1,4-benzodiazepine motif. For example, the 1,4-benzodiazepine-2,5-dione skeleton is considered to be of a special interest as established drug-like scaffold in recent medicinal chemistry, as illustrated in figure XI.1. Besides the important seven-membered benzodiazepine privileged structures, the corresponding eight membered homologue could be potentially interesting for drug discovery purposes. Examples of these biologically active benzodiazocines are known (see figure XI.1), but are clearly underrepresented, probably as a consequence of the known difficulties to synthesize such challenging medium-ring skeletons.

H O O H O H O N HO N N HN Cl I N MeO N O COOH N N O O HO

HDM2 antagonist (RK-1441B) antibiotic (+)-CGP 48506 muscle relaxant XI.1 XI.2 XI.3 XI.4 Figure XI.1 Examples of bio-active compounds built around a central privileged structure. Left: 1,4-benzodiazepine-2,5-dione derivative XI.1 and XI.2. Right: 1,5-benzodiazocine derivative XI.3 and XI.4.

To be able to maximally exploit a given privileged structure in diverse drug discovery programs, a general and straightforward synthesis methodology to the skeleton would be advantageous, in which a high structural side chain diversity could be introduced. To this end, a solid phase synthesis

170

Part 2: Results and Discussion approach considered to be a useful tool (combinatorial chemistry), which could potentially synthesize a broad range of analogues using sets of modular building blocks, and has shorten the cost and time.

XI.2. AIM AND OBJECTIVES

The aim of this PhD thesis is to synthesize libraries of structurally diverse compounds on the basis of a 1,5-benzodiazocine-2,6-dione skeleton (figure XI.2, general structure of derivatives: XI.5), through a solid-phase chemistry-based approach. These compounds have only limitedly been reported in literature.

R1 O 10 N 10c 2 R 9 1 3 2 R5 4 8 6 5 R 7 6a N 3 O R4 XI.5

Figure XI.2 Representative structure for the 1,5-benzodiazocine-2,6-dione skeleton

The specific aim of this project are 1) an initial optimization of a previously explored synthetic route, 2) validation of the synthetic route to 3- and 4-substituted analogues and 3) the synthesis of more challenging 3,3-disubstituted-1,5-benzodiazocine-2,6-diones. Moreover, the required building blocks (β-amino acids) will have to be synthesized in-house as they are generally not commercially available.

XI.3. RESULTS AND DISCUSSION

XI.3.1. General synthesis strategy

Retrosynthesis of the intended 1,5-benzodiazocine-2,6-dione skeleton results in two basic building blocks: anthranilic acid derivatives XI.8 and β-amino acids XI.9; which can be connected via two amide bonds.

171

Part 2: Results and Discussion

NHFmoc HOOC

R4 anthranilic acids

XI.8 O H + N lactam R1 O R2 O NH2 amide bond O R2 formation formation R4 O N O NH N R2 R R R R O R 1 3 1 3 3 R4 XI.5 XI.6 XI.7 -amino acid Mitsunobu-Fukayama coupling and N- alkylation (R2OH)

O R2 OH + HO NHFmoc

R1 -amino acid XI.10 XI.9

Scheme XI.1 Retrosynthesis for target benzodiazocinedione XI.5.

XI.3.2 Solid phase synthesis of a model 1,5-benzodiazocine-2,6-dione

In order to verify and optimize the earlier developed methodology3, 5-benzyl-3,4-dihydro-1,5- benzodiazocine-2(1H),6(5H)-dione XI.11 has been chosen as model target compound. The synthesis involved Wang resin-loading of Fmoc β-alanine XI.13 (scheme XI.2) followed by removal of the fluorenylmethoxycarbonyl moiety using 4-methylpiperidine in DMF, N-mono- benzylation through a Mitsunobu-Fukayama alkylation sequence, coupling with the symmetrical anhydride of N-Fmoc-anthranilic acid and Fmoc-removal to form resin-bound dipeptide XI.18. Previous trials to cyclize the aminoester using acid-, base-, or nucleophilic catalysts failed, while the use of strong bases (KOtBu, NaH) caused the formation of an elimination product arising from acidic α-protons3. Therefore, cleavage of the dipeptide X.19 from the resin was performed with TFA a range of cyclization conditions was tried. Among the tested conditions, cyclization could be achieved in solution using ClCOOMe or by reaction with solid-support-bound DCC-coupling reagent (75% isolated yield for both). In the latter case, the formation of 2 % of 16-membered ring

172

Part 2: Results and Discussion next to 8-membered ring was obseved in addition to the occurrence of an O→N acyl migration of the DCC resin-activated carboxylic acid. However, the easier work up of the DCC resin (filteration) convinced us to apply these conditions in the next libraries

O O NHFmoc a c, d, e OH + HOOC O NHR O NH XI.12 XI.13 Bn XI.16 R= Fmoc XI.14 b R= H XI.15 f

O O O NHR H O O NH2 N h HO N g O N 75% Bn Bn 88% from XI.14 N O Bn XI.19 R= Fmoc XI.17 b R= H XI.18 XI.11

a) 1) 2 eq XI.13, 2 eq DIC, CH2Cl2, 0.2 eq DMAP, 24h. 2) Ac2O/ DIPEA/DCM 1/1/3, 2 x 2 h. b) 20% 4- methylpiperidine in DMF 2 x 20 min. c) 5 eq o-NsCl, 10 eq collidine, CH2Cl2, 2 x 1 h. d) 10 BnOH, 5 eq PPh3, 5 eq DIAD, DCE, 3 x 2 h. e) 2.5 eq DBU, 5 eq 2-mercaptoethanol, DMF, 2 x 30 min. f) 10 eq Fmoc-anthranilic acid, 5 eq DIC, DCM/DMF 9/1, 24 h. g) TFA/H2O 95/5, 2 x 1 h. h) polystyrene-bound-DCC, DCM, 1h.

Scheme XI.2 Synthesis of 5-benzyl-3,4-dihydro-1,5-benzodiazocine-2(1H),6(5H)-dione XI.11.

XI.3.3 Synthesis of 3-substituted-1,5-benzodiazocine-2,6-diones

XI.3.3.1 Synthesis of N-Fmoc-anthranilic acid building blocks

Fmoc protection of commercially available 2-aminobenzoic acids with Fmoc-OSu delivered six N-Fmoc-2-aminobenzoic acid derivatives XI.21-XI.25 (scheme XI.3). Attempts to the synthesis of 5-phenyl anthranilic acid turned out to be less straightforward. Neither Suzuki cross-coupling reaction of 5-iodoisatine with phenylboronic acid and subsequent Baeyer-Villiger oxidation, nor ortho-lithiation of N-Boc-4-phenylaniline acid provided the desired product in appropriate yield.

173

Part 2: Results and Discussion

3 3 NHFmoc 2 NH2 4 2 4 i) 1 eq NaOH, H2O, rt, 5 min 1 R 1 R 5 5 ii) 1 eq NaHCO3, 1 eq Fmoc-OSu, COOH 6 COOH 6 THF/H2O 2/1, r.t, o.n

2-aminobenzoic acid Fmoc-2-aminobenzoic acid XI.20 R= H R= H XI.21 R=5-bromo R=5-bromo XI.22 R=5-methoxy R=5-methoxy XI.23 R=5-methyl R=5-methyl XI.24 R=6-fluoro R=6-fluoro XI.25 R=4-trifluoromethyl R=4-trifluoromethyl XI.26

Scheme XI.3 Synthesis of N-Fmoc-2-aminobenzoic acid derivatives.

XI.3.3.2 Synthesis of N-Fmoc-3-amino-2-alkylpropionic acid building blocks

Although in this PhD project we used racemic β2-amino acids, optically pure derivatives could also be applied. However, general methods for their preparation are limited, but are reported from linear or cyclic β-alanine derivatives4,5 or by homochiral aminomethylation of 2-alkylacetic acid enolates6.

N-Fmoc-2-alkyl-3-aminopropionic acid building blocks were successfully prepared (as depicted in scheme XI.4) starting from the appropriate aldehydes and methyl cyanoacetate which were reacted via a Knoevenagel condensation followed by reduction using Pd/C-catalyzed hydrogenation (R=isopropyl) or NaCNBH3/AcOH to give compounds XI.29. Chemoselective reduction of the cyano group using NiCl2.6H2O/NaBH4 and in situ Boc-protection gave protected 2 β -amino acid intermediates XI.30. Hydrolysis of the ester moiety in those compounds was accomplished using NaOH, while the N-terminus was reprotected as fluorenylmethyl carbamate to the Fmoc-protected amino acids using Fmoc-OSu/Na2CO3. The building blocks were obtained in good overall yields.

Intended compounds XI.36 and XI.37 (figure XI.3) could not be obtained successfully as bromobenzyl derivative XI.37 was contaminated with a considerable amount (11%) of the de- halogenated analogue, while for XI.36, the final N-protection sequence could not be achieved because of total decomposition in the acidic medium used for Boc-removal.

174

Part 2: Results and Discussion

g (R= isopropyl)

O Boc COOMe a NC COOMe b NC COOMe c N d H H R (R=2-naphthyl or R 4-chlorophenyl R R XI.27 XI.28 XI.29 XI.30

Boc COOH e COOH f Fmoc COOH N HCl.H2N N H H R R overall yield R XI.33: R = isopropyl 55%. XI.31 XI.32 XI.34: R = 2-naphthyl 34%. XI.35: R = 4-chlorophenyl 50%.

a) 1 eq methyl cyanoacetate, 0.1 eq AcOH, 0.04 eq piperidine, dioxane, r.t., o.n. b) 1.1 eq NaCNBH3, 0.4 M AcOH, MeOH, r.t, 1h to o.n. c) 0.1 eq NiCl2.6H2O, 7 eq NaBH4, 2 eq Boc2O, MeOH, 0C to r.t., o.n. d) 4 eq NaOH (H2O/MeOH 2/1), r.t to 50° C, 20 min to o.n. e) Dioxane / concentrated HCl 9/1, r.t, 7h to o.n. f ) 3 eq Na2CO3,1 eq Fmoc-OSu, THF/H2O (2/1), r.t., o.n. g) 1 eq methyl cyanoacetate, 0.1 eq AcOH, 0.04 eq piperidine, dioxane, Pd/C 5% wt, H2 (2 bar), r.t, 2 h. Scheme XI.4 Synthesis of N-Fmoc-2-alkyl-3-aminopropionic acid.

COOH COOH FmocHN FmocHN

O Br XI.36 XI.37

Figure XI.3 Intended compounds which could not be obtained.

XI.3.3.3 Alcohols

A series of primary alcohols were tested during the optimization of model compound, and a set of successful alcohols was chosen to decorate the N-5 position in the target 3-substituted-1,5- benzodiazocine-2,6-diones.

XI.3.3.4 Library Synthesis

The synthetic route towards 3-substituted-1,5-benzodiazocine-2,6-diones could be performed using the procedure developed for the model 1,5-benzodiazocine-2,6-dione.

175

Part 2: Results and Discussion

O O a c, d, e OH O NHR O NH

R1 R1 R2 XI.12 R= Fmoc XI.38 XI.40 R= H XI.39 b f

O O O NHR H O O NH2 N g R1 h HO N O N R R1 R2 3 R1 R2 N R3 R3 O R2 XI.43 R= Fmoc XI.41 XI.44 b R= H XI.42

a) 1) 2 eq XI.33-XI.35, 2 eq DIC, CH2Cl2, 0.2 eq DMAP, 24h. 2) Ac2O/ DIPEA/DCM 1/1/3, 2 x 2 h. b) 20% 4- methylpiperidine in DMF 2 x 20 min. c) 5 eq o-NsCl, 10 eq collidine, CH2Cl2, 2 x 1 h .d) 10 eq alcohol, 5 eq PPh3, 5 eq DIAD, DCE, 3 x 2 h. e) 2.5 eq DBU, 5 eq 2-mercaptoethanol, DMF, 2 x 30 min. f) 10 eq Fmoc-anthranilic acid XI.21-XI.26, 5 eq DIC, DCM/DMF 9/1, 24 h. g) TFA/H2O 95/5, 2x1h. h) polystyrene-bound-DCC, DCM, 1h. Scheme XI.5 Synthetic route towards 3-substituted-1,5-benzodiazocine-2,6-diones XI.44.

The overall yields of solid-phase synthesis of ring-closing precursors are moderate to good (figure XI.4). Cyclization yields using polystyrene-bound DCC were low to moderate, in agreement with expected O→N acyl migration side reactions.

176

Part 2: Results and Discussion

O O H O H H N N N

N N MeO N O F O O XI.46 O XI.45 O XI.47 SPS: 65% SPS% 53% SPS : 31% CYC: 67% CYC: 55% CYC: 30 % O O H O H H N N N

N N N O O O

XI.48 XI.49 XI.50 SPS: 62% OMe SPS 38% SPS: 37% CYC: 41% CYC: 44% CYC: 80% O O H H H O N N N

Br N MeO N Me N O O O

XI.51 XI.52 XI.53 F SPPS: 63% SPS 35 % SPPS: 62% CYC: 63% CYC: 21% CYC: 41% Cl Cl Cl Cl

O O O H O H H N H N N N

Br N MeO N Br N N O O O O O N XI.54 XI.55 XI.56 O XI.57 SPS: 50% SPS: 69% SPS: 30% SPS: 87% CYC:27% CYC: 39% CYC: 43% CYC:25%

Figure XI.4 Synthesized library of 3-substituted-1,5-benzodiazocine-2,6-diones.

XI.3.4 SYNTHESIS OF 4-SUBSTITUTED-1,5-BENZODIAZOCINE-2,6-DIONES

Based on the proposed solid-phase synthesis strategy, essential building blocks consist of N-Fmoc- anthranilic acids and N-Fmoc-3-amino-3-alkylpropionic acids.

177

Part 2: Results and Discussion

XI.3.4.1 N-Fmoc-anthranilic acid building blocks

The different N-Fmoc-anthranilic acids were prepared (scheme XI.3).

XI.3.4.2 Synthesis of N-Fmoc-3-amino-3-alkylpropionic acid building blocks

In this study, only racemic β-amino acid building blocks were used. However, enantiopure β3- amino acids are available by preparation from α-amino acids using Arndt-Eistert homologation7, but because of security risks associated with diazomethane needed in the reaction; an alternative method via nucleophilic substitution with CN- 8 may be preferred to use it in a large scale production.

N-Fmoc-3-amino-3-alkylpropionic acid building blocks were successfully prepared by treating the commercially available β3-amino acids XI.58 with equivalent amounts of Fmoc-OSu in the presence of Na2CO3.

O R O R 2 eq Na2CO3, 1 eq Fmoc-OSu, HO NH HO NHFmoc 2 THF/H2O 2/1, r.t, o.n XI.58 XI.59 R= Me, yield=90% XI.60 R=i-Bu, yield=87% XI.61 R=Bn, yield=84%

Scheme XI.6: Synthesis of N-Fmoc-3-alkyl-3-aminopropionic acid derivatives.

XI.3.4.3 Library Synthesis

The synthetic route towards 4-substituted-1,5-benzodiazocine-2,6-dione ran parallel to 3- substituted-1,5-benzodiazocine-2,6-dione derivatives, applying the N-Fmoc-β3-amino acids in the proposed synthetic route. Due to steric reasons, a double coupling procedure of N-Fmoc anthranilic acids was found to be necessary. The overall moderate to good yields (depicted in figure XI.5) for the solid-phase synthesis of ring-closing precursors were comparable to the ones obtained for the 3-substituted-1,5-benzodiazocine-2,6-diones. Cyclization yields using polystyrene-bound DCC were low to moderate, which are attributed to O→N acyl migration side reactions.

178

Part 2: Results and Discussion

O O O H H H H O N N N N

MeO N N N MeO N O O O O H O N XI.62 XI.63 XI.64 XI.65 O SPS: 77% SPS: 47% SPS: 57% CF SPS: 41% CYC: 65% CYC: 45% CYC: 23% 3 CYC:18%

O O H O H H N N N

Br N N N O O O XI.66 XI.67 XI.68 SPS: 76% SPS: 48% SPS: 44% CYC: 46% CYC: 40% CYC: 31% O O O H H H O H N N N N F3C

Br N N N N O O O O F XI.69 XI.70 XI.71 XI.72 SPS: 56% OMe SPS: 56% SPS: 98% SPS: 73% CYC: 57% CYC: 24% CYC: 45% CYC: 45% Figure X.5 Synthesized library of 4-substituted-1,5-benzodiazocine-2,6-diones.

XI.3.5 Synthesis of 3,3-disubstituted-1,5-benzodiazocine-2,6-diones

Based on the proposed solid phase synthesis strategy, essential building blocks consist of N-Fmoc- anthranilic acids and N-Fmoc-3,3-dialkyl-3-aminopropionic acids. The synthesis of 3,3-dimethyl- 1,5-benzodiazocine-2,6-diones was previously demonstrated at our laboratory; using simple N- Fmoc-3,3-dimethyl-3-aminopropionic acid building block. To further validate this synthetic strategy a more complex model building block XI.73 was successfully prepared in moderate overall yield (synthetic route is depicted in scheme XI.7).

Using this model β2,2-amino acid building block XI.73, the solid-phase synthesis of model benzodiazocinedione XI.87 was investigated (detailed reaction conditions is depicted in scheme XI.8).

179

Part 2: Results and Discussion

O a NC COOMe b NC COOMe c NC COOMe H Me 88% 92% 78%

XI.74 XI.75 XI.76 XI.77 Boc COOMe Boc COOH COOH d N e N f HCl.H2N g H Me H Me Me 60% 96% 99% 44%

XI.78 XI.79 XI.80

Fmoc COOH N H Me

XI.73 16% overall yield, 7 steps

a) 1 eq methyl cyanoacetate, 0.1 eq AcOH, 0.04 eq piperidine, dioxane, r.t., o.n. b) 1.1 eq NaCNBH3, 0.4 M AcOH, MeOH/CH2Cl2 5/1, r.t, 1h. c) 5 eq MeI, 2 eq NaOMe, THF, 1M, 1h. d) 0.1 eq NiCl2.6H2O, 7 eq NaBH4, 2 eq Boc2O, MeOH, 0C to r.t., o.n. e) 4 eq NaOH, (H2O/MeOH 2/1), r.t to 50° C, 2h. f) Dioxane / concentrated HCl 9/1, r.t, 7h. g)i) 2 eq TMSiCl, 1 eq DIPEA, CH2Cl2, reflux 6 h. ii) 2 eq DIPEA, 1 eq FmocCl, 0 C-r.t, o.n. Scheme XI.7 Synthetic route to β2,2-amino acid building block XI.73.

O O a c, d, e O NH OH O NHR 2 Me Me

XI.12

XI.83 b R= Fmoc XI.81 R= H XI.82

O O NH2 O O NHR H O N Me h HO N g O N Me Me 60 % Me 94 % Me N O Me XI.87

XI.86 b R= Fmoc XI.84 R= H XI.85

a) 1) 2 eq XI.73, 2 eq DIC, CH2Cl2, 0.2 eq DMAP, 24h. 2) Ac2O/ DIPEA/CH2Cl2 1/1/3 , 2x2 h. b) 20% 4- methylpiperidine in DMF, 2 x 20 min. c) 5 eq o-NsCl, 10 eq collidine, CH2Cl2, 2 x 1 h. d) 10 eq MeOH, 5 eq Ph3P, 5 eq DIAD, DCE, 3 x 2 h. e) 2.5 eq DBU, 5 eq 2-mercaptoethanol, DMF, 2 x 30 min. f) 10 eq Fmoc-anthranilic acid, 5 eq DIC, CH2Cl2/DMF 9/1, 24 h. g) TFA/H2O 95/5, 2 x 1 h. h) polystyrene-bound-DCC, CH2Cl2, 1h.

Scheme XI.8 Solid-phase synthetic route towards XI.87.

180

Part 2: Results and Discussion

Unfortunately, the anticipated direct cyclization/release approach from precursor XI.85 using KOtBu did not yield the expected benzodiazocinedione. Alternatively, the solution-phase cyclization using resin-bound DCC delivered the target bislactam. In conclusion, it seems the increased steric congestion in the cyclization precursor hampers the general application of the KOtBu-mediated cyclization methodology as used before for 3,3-dimethylated-1,5- benzodiazocine-2,6-diones.

XI.3.5 Conformational and Modeling study

A modeling experiment clearly show a ground state twist-boat conformation for both of the synthesized two libraries (3-substituted and 4-substituted-1,5-benzodiazocine-2,6-diones). The presence of two conformational states in the synthesized 1,5-benzodiazocine-2,6-diones was clear from the corresponding complex 1H NMR and 13C NMR spectra. Higher temperature 1H NMR experiment was performed for selected members of the two libraries revealed that for presence of an N-5 substituent increases the energy barriers for conformational processes; at room temperature, compound XI.74 is conformationally locked and exists as a mixture of uninterchangeable chiral conformers (as the only compound clearly seen in LCMS as two separable peaks) indicating a very high energy barrier for ring-interconversion. Unlike compound XI.75 high temperature 1H NMR experiment shows coalescence in peaks that leads to calculation of energy barrier at coalescence. Thermodynamic equilibrium present between two conformers can be concluded

H O H O N N

N N F O O O O XI.74 XI.75

XI.4 CONCLUSION AND FUTURE PERESPECTIVE

The initial objectives of this project have been reached: the initially chosen model compound could be synthesized after optimization of the proposed solid-phase synthetic route and small libraries of 3-and 4-substituted-1,5-benzodiazocine-2,6-diones were synthesized in good to moderate yields. The more challenging quaternary carbon on the position 3 of the ring system could be introduced, albeit not via the proposed cyclization/release strategy.

181

Part 2: Results and Discussion

The established solid-phase buildup and solution-phase cyclization methodologies can be applied in the future for the construction of further libraries of pharmaceutically relevant analogues. In this regard, further investigation on the possibilities of C3,C4-disubstitution could be useful.

182

Part 2: Results and Discussion

Refreneces

1 Evans, B. E.; Rittle, K. E.; Bock, M. G.; Di-Pardo, R. M.; Freidinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.; Anderson, P. S.; Chang, R. S.; Lotti, V. J.; Cerno, D. J.; Chen, T. B.; Kling, P. J.; Kunkel, K. A.; Springer, J. P.; Hirshfield, J. J. Med. Chem. 1988, 31(12), 2235–2246.

2 Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev. 2001, 46(1- 3), 3-26.

3 Caroen J., Ontwikkeling van een vastefasesynthesestrategie voor 1,2,3,4,5,6-hexahydro-1,5- benzodiazocine-2,6-dionen voor toepassing in combinatorische bibliotheken, (Development of a solid-phase synthesis strategy for 1,2,3,4,5,6-hexahydro-1,5-benzodiazocine-2,6-diones for application in combinatorial libraries) 2012. PhD dissertation, UGent.

4 Juaristi, E.; Quintana, D.; Balderas, M.; García-Pérez, E. Tetrahedron Asymm. 1996, 7, 2233- 2246.

5 Seebach, D.; Boog, A.; Schweizer, W.B. Eur. J. Org. Chem. 1999, 335-360.

6 Tessier, A.; Lahmar, N.; Pytkowicz, J.; Brigaud, T. J. Org. Chem. 2008, 73, 3970-3973.

7 Podlech, J.; Seebach, D. Liebigs Ann. 1995, 1217-1228.

8 Caputo, R.; Cassano, E.; Longobardo, L.; Palumbo, G. Tetrahedron 1995, 51, 12337-12350.

183

Part 2: Results and Discussion

XII NEDERLANDSE SAMENVATTING

In dit doctoraatswerk werd de vastefasesynthese van structureel diverse 1,5-benzodiazocine-2,6- dionen beoogd. Dit relatief onverkende skelet is potentieel interessant vanuit farmaceutisch perspectief, aangezien het een duidelijke verwantschap vertoont met de populaire benzodiazepine bevoorrechte structuren, en daardoor een bruikbare scaffold kan betekenen voor de ontwikkeling van nieuwe geneesmiddelen inwerkend op een brede waaier aan diverse biochemische doelwitstructuren.

Om een zo algemeen mogelijke vlotte toegangsweg te ontwikkelen tot een breed gamma aan gesubstitueerde analogen, werd een vastefasesynthesemethodologie vooropgesteld, aangezien dit door het gebruik van modulaire bouwstenen en de eenvoudige intermediaire opzuiveringsstappen de mogelijkheid biedt tot automatisering.

Schema XII.1 Algemene syntheseroute tot de beoogde 1,5-benzodiazocine-2,6-dionen.

De syntheseroute (schema XII.1) houdt in het algemeen de opeenvolgende koppeling van een beta- aminozuurbouwsteen op Wanghars in, waarna de ontschermde primaire aminegroep wordt mono-

184

Part 2: Results and Discussion gealkyleerd in een zogenaamde Mitsunobu-Fukuyama sequentie met behulp van commercieel verkrijgbare alcoholen. Koppeling van Fmoc-anthranilzuurbouwstenen met daaropvolgende N- ontscherming levert de ringsluitingsprecursoren die na harsafsplitsing gecycliseerd worden met behulp van een harsgebonden carbodiimidereagens.

In een eerste deel werd deze reeds vroeger aan ons laboratorium verkende vastesyntheseroute toegepast en geoptimaliseerd voor een modelverbinding (XII.8, R1=R2=R4=H, R3=Bn), leidend tot de bepaling van algemeen bruikbare reactiecondities om toe te passen voor verdere bibliotheeksynthesen.

In een tweede deel werden deze reactie-omstandigheden gebruikt voor de synthese van 3- gesubstitueerde benzodiazocines, door gebruik te maken van een set geschikte β2- aminozuurbouwstenen. Deze bouwstenen werden gesynthetiseerd uitgaande van methylcyanoacetaat en verschillende aldehyden, via initiële Knoevenaegel condensatie. Toepassing van de achtstapsvastefasesynthese leverde een bibliotheek van 13 cyclisatieprecursoren XII.7 in matig tot goede rendementen (30-87%) die werden gecycliseerd tot de overeenkomstige 1,5-benzodiazocine-2,6-dionen XII.8 (21-80%).

Vervolgens werd de methodologie eveneens toegepast op de synthese van een serie 4- gesubstitueerde benzodiazocines. Hierbij werd gebruik gemaakt van commercieel verkrijgbare β3- aminozuurbouwstenen, die beschermd werden als fluorenylmethylcarbamaat. De hogerontwikkelde omstandigheden leidden op deze manier tot een bibliotheek van 11 cyclisatieprecursoren XII.7 (41-98% rendement) die succesvol konden worden gecycliseerd tot de bedoelde 1,5-benzodiazocine-2,6-dionen XII.18 (18-65% rendement).

In een laatste doelstelling werden eveneens verbindingen met een 3,3-disubstitutiepatroon beoogd. Vroeger onderzoek aan ons laboratorium had aangetoond dat de synthese van 3,3- dimethylanalogen moglijk is via directe cyclisatie van de harsgebonden precursoren na behandeling met KOtBu. Hiertoe werd een model β2,2-aminozuurbouwsteen gesynthetiseerd, die werd omgezet via de vastefasesynthesestrategie tot het cruciale harsgebonden intermediair XII.9 (schema XII.2). Pogingen om de directe cyclisatie met gelijktijdige afsplitsing van het gewenste achtringskelet XII.11 te bewerkstelligen, leverden echter geen product op, zodat de cyclisatie

185

Part 2: Results and Discussion diende te worden uitgevoerd met harsgebonden carbodiimidereagens na afsplitsing van de precursor XII.10 in oplossing.

Schema XII.2 Synthese van 3,3-digesubstitueerde modelverbinding XII.11.

De complexe NMR spectra van de gesynthetiseerde 1,5-benzodiazocine-2,6-dionen tonen de aanwezigheid aan van atropoisomerie, een potentieel belangrijk fenomeen in farmaceutische toepassingen. Ondersteunende modelling experimenten en vergelijking met literatuurvoorbeelden leert dat de verbindingen twee geïnverteerde boot-type conformaties aannemen, die kunnen interconverteren via een relatief hoge energiebarrière. Voor 4,5-dimethyl-1,5-benzodiazocine-2,6- dion XII.12 (figuur XII.1) kon uit een serie 1H NMR-experimenten bij verschillende temperaturen ≠ op basis van waargenomen coalescentieverschijnselen (Tcoal~100°C) een barrière ΔG van 77.6 kJ/mol geschat worden, wat wijst op een vlotte boot/boot-interconversie bij kamertemperatuur. Bij analogen met complexere substituenten kan in de NMR spectra geen piekverbreding meer worden waargenomen (T=130°C), wat wijst op hogere interconversiebarrières. Voor verbinding XII.13 konden bovendien (als enige in de aangemaakte bibliotheken) in LCMS-analysen beide conformeren als gescheiden pieken worden waargenomen, wat er op wijst dat deze molecule als twee stabiele atropoïsomeren voorkomt.

186

Part 2: Results and Discussion

Figuur XII.1 Structuur van verbindingen XII.12 en XII.13, gebruikt voor een conformationele studie.

In elk geval is verder onderzoek wenselijk om een meer gedetailleerd inzicht te verkrijgen in het conformationeel gedrag van deze potentieel farmaceutisch interessante structuren.

187

PART 3: EXPERIMENTAL PROCEDURES

Part 3: Experimental Procedures

XIII.1 INSTRUMENTATION AND METHODS

XIII.1.1 Solvents and reagents

All commercial solvents and reagents were used as received, unless otherwise noted. Reactions in solution were performed with freshly distilled solvents, reactions on solid phase were performed with freshly distilled solvents or HPLC quality. Washing of resins was performed with solvents of HPLC grade. Dichloromethane, 1,2-dichloroethane, triethylamine and diisopropylethylamine were dried on calcium chloride. THF was dried on sodium and benzophenone.

XIII.1.2 Purification

Column chromatography was performed standard with Grace Division LC 60Å silica (60-200 μm) or (in case of difficult separation) with Rocc silica 60Å (40-60 μm). The eluent consisted of technical solvents for large scale purification and of HPLC solvents for small scale purifications.

Preparative HPLC-purifications of more than 10 mg crude product were performed on a Phenomenex Luna C18 column (5μm, 250 x 21,20 mm), making use of a Kontron 422 type HPLC- pump and a MELZ lcd 312 RI-detector. For purifications of less than 10 mg product, an adjusted Phenomenex Luna C18 column (5μm, 250 x 10,00 mm) was applied on the same system.

XIII.1.3 Characterization

Reactions in solution were monitored using Thin Layer Chromatography (TLC). The Rf values were determined on Machery-Nagel SIL-G25 U254 TLC-plates, by using UV-light (254 nm) or by developing them with a suitable staining reagent (a KMnO4 solution in water, cerium molybdate/Hanessian's staining or ninhydrine solution).

Reactions on solid phase were monitored by LC-MS after cleaving off a small amount of product from the solid support by treatment with an appropriate cleavage reagent (TFA/H2O 9/1). The analyses were performed on a VL type Agilent 1100 LC/MS system, charged with a Phenomenex Luna C18 column (methods A and B) or with a Kinetex C18 column (methods C and D). Reported retention times are obtained by method A, unless specified otherwise. A 5mM solution of NH4OAc in water (solvent A) and acetonitrile (solvent B) were used as eluents on this system. The gradients are mentioned below.

188

Part 3: Experimental Procedures

Method A

Time Solvent A Solvent B 0 min 100 0 2 min 100 0 17 min 0 100 22 min 0 100 22.5 min 100 0 30 100 0

Method B Time Solvent A Solvent B 0 min 80 0 2 min 80 0 32 min 0 80 37 min 0 80 37.5 min 80 0 40 80 0

Method C Time Solvent A Solvent B 0 min 80 20 2 min 80 20 32 min 20 80 37 min 20 80 37.5 min 80 20 40 80 20

189

Part 3: Experimental Procedures

Method D

Time Solvent A Solvent B

0 min 100 50

2 min 100 50

32 min 50 100

37 min 50 100

37.5 min 100 50

40 100 50

Method E

Time Solvent A Solvent B

0 min 80 50

2 min 80 50

32 min 50 80

37 min 50 80

37.5 min 80 50

40 80 50

UV spectra were recorded using a Hitachi U-2010 UV-VIS Spectrophotometer or Varian Cary 3E UV-VIS Spectrophotometer. NMR analyses were performed on a Bruker Avance 300, 400 or a Bruker DRX500. 1H NMR measurements were performed at 300, 400 or 500 MHz; 13C NMR measurements at 75, 100 or 125 MHz on the above-mentioned spectrometers. Chemical shifts are reported in ppm, using the residual solvent signal as reference value (CDCl3: 7.26 ppm/77.00 ppm,

190

Part 3: Experimental Procedures

DMSO-d6: 2.50 ppm/39.51 ppm), acetone-d6: 2.05/29.84 ppm and D2O: 4.79 ppm). Melting points were recorded on a Kofler melting apparatus.

XIII.1.4 Equipment

Small scale solid phase reactions (< 50 mg resin) were performed in small glass vials using a Selecta Vibromatic whristshaker. Larger scale reactions were performed in homemade screw- capped glass tubes equipped with a glass fritted filter and filtration system for the vacuum pump.

191

Part 3: Experimental Procedures

XIII.2 SYNTHESIS OF FMOC-PROTECTED ANTHRANILIC ACID DERIVATIVES

XIII.2.1 General procedure

NH 2 i) 1eq NaOH, H2O, r.t, 5 min NHFmoc

ii) 1eq NaHCO3, 1eq Fmoc-OSu COOH x COOH x THF/H2O 2/1, r.t, o.n

To a solution of anthranilic acid (72.9 mmol, 1 eq) in 1M NaOH (72.9 mmol, 1 eq) is added THF

(73 ml) and Fmoc-OSu (72.9 mmol, 1 eq). After 5 min, THF (72 ml) and NaHCO3 (72.9 mmol, 1 eq) is added and the resulting mixture is stirred overnight at room temperature. The reaction mixture is acidified using 12 M HCl until pH=1-2 and after adding (100 ml) H2O, the aqueous phase is extracted three times with ethyl acetate. The combined organic layers are dried with anhydrous magnesium sulfate, the drying agent filtered and the volatiles removed under reduced pressure. The crude solid is purified by recrystallization.

XIII.2.2 N-(9-FLUORENYLMETHOXYCARBONYL)-2-AMINOBENZOIC ACID VI.7

Recrystallization solvent: EtOAc

Yield: 87%; white fluffy powder, 22.8 g from 10 g anthranilic acid

H M.P.: 226 °C N O

O Molecular Formula: C22H17NO4 COOH Molecular weight: 359.37 g mol-1

LC-MS: Peak at 4.9 min; ES-MS negative mode [m/z (fragment, intensity)]: 358.1 (M-H+, 61), - - 162.0 (O=C=N-Ph-COO , 100), 136.0 (H2N-Ph-COO , 47).

1 H NMR: (500 MHz, DMSO-d6): δ 10.83 (s, 1H), 8.16 (br. d, J = 7.9 Hz, 1H), 7.98 (d, J = 7.9 Hz, 1H), 7.93 (d, J = 7.5 Hz, 2H), 7.70 (d, J = 7.5 Hz, 2H), 7.58 (t, J = 7.9 Hz, 1H), 7.44 (t, J = 7.4 Hz, 2H), 7.35 (J = 7.4 Hz, 2H), 7.12 (t, J = 7.6 Hz, 1H), 4.50 (d, J = 6.7 Hz, 2H), 4.37 (t, J = 6.0 Hz, 1H) ppm.

192

Part 3: Experimental Procedures

13 C NMR: (125 MHz, DMSO-d6): δ 170.3 (C), 153.2 (C), 144.2 (C), 141.4 (C), 141.3 (C), 134.8 (CH), 131.8 (CH), 128.3 (CH), 127.7 (CH), 125.5 (CH), 122.6 (CH), 120.8 (CH), 118.9 (CH),

116.3 (C), 66.8 (CH2), 47.0 (CH) ppm. IR (HATR): 3320 (m, sharp), 2500-3300 (m, broad), 3066 (w), 2988 (w), 2948 (w), 1740 (s), 1665 (s), 1602 (m), 1586 (s), 1535 (m), 1523 (s), 1468 (m), 1452 (m), 1399 (m), 1327 (w), 1300 (m), 1258 (s), 1212 (s), 1166 (m), 1152 (w), 1096 (m), 1068 (w), 1052 (s), 1043 (w), 1002 (w), 953 (w), 935 (w), 897 (m), 862 (w), 794 (w), 766 (s), 744 (m), 726 (w), 683 (m), 668 (w), 650 (w), 622 (w), 588 (w), 553 (w), 538 (m) cm-1.

TLC: Rf = 0.38 (Dichloromethane/MeOH 70/30).

XIII.2.3 N-(9-FLUORENYLMETHOXYCARBONYL)-2-AMINO-5-BROMOBENZOIC ACID VI.8

Recrystallization solvent: EtOAc

Yield: 85 % (white powder), 5.1 g from 3 g 2-amino-5- bromobenzoic acid H N O M.P.: 256 °C O Br COOH Molecular Formula: C22H16BrNO4

Molecular weight: 438.27 g mol-1

LC-MS: Peak at 5.3 min; ES-MS negative mode [m/z (fragment, intensity)]: 438.0 (M(81Br)-H+, 100), 436.0 (M(79Br)-H+, 100).

81 + + HR-MS (ESI): C22H17 BrNO4 [M+H ]: Calculated 438.0341 Found 438.0352.

1 H NMR: (300 MHz, DMSO-d6): δ 10.69 (s, 1H), 8.09 (d, J = 9.0 Hz, 1H), 8.02 (d, J = 2.0 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.73 (br d, J = 8.8 Hz, 1H), 7.67 (d, J = 7.3 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 4.49 (d, J = 6.6 Hz, 2H), 4.34 (t, J = 6.4 Hz, 1H) ppm. 13 C NMR: (75 MHz, DMSO-d6): δ 168.3 (C), 152.5 (C), 143.5 (C), 140.8 (C), 140.0 (C), 136.6 (CH), 133.2 (CH), 127.7 (CH), 127.1 (CH), 125.0 (CH), 120.7 (CH), 120.2 (CH), 118.0 (C), 113.4

(C), 66.4 (CH2), 46.4 (CH) ppm.

193

Part 3: Experimental Procedures

IR (HATR): 3429 (w), 3333 (vw), 3042 (vw), 1733 (m), 1672 (m), 1595 (w), 1578 (m), 1517 (m), 1461 (w), 1447 (w), 1409 (w), 1387 (m), 1310 (w), 1287 (vw), 1242 (s), 1215 (s), 1189 (m), 1158 (w), 1100 (w), 1092 (w) , 1076 (w), 1040 (m), 1009 (vw), 975 (vw), 939 (vw), 893 (vw), 866 (vw), 837 (w), 814 (vw), 790 (w), 767 (vw) 756 (w), 733 (m), 722 (w), 699 (m), 675 (w), 645 (w) cm-1.

TLC: Rf = 0.32 (Dichloromethane/MeOH 70/30).

XIII.2.4 N-(9-FLUORENYLMETHOXYCARBONYL)-2-AMINO-5-METHOXYBENZOIC ACID VI.9

Recrystallization solvent: aqueous MeOH.

Yield: 63 %; (off-white cotton powder), 4.0 g from 2.5 g 2-

H amino-5-methoxybenzoic acid. N O M.P.: 240 °C O MeO COOH Molecular Formula: C23H19NO5

Molecular weight: 389.41 g mol-1.

LC-MS: Peak at 4.8 min; ES-MS negative mode [m/z (fragment, intensity)]: 388.0 (M-H+, 60), + 192.0 (M-fluorenylmethanol-H , 100), 166.1(M-dibenzofulvene-CO2, 58).

+ + HR-MS (ESI): C23H20NO5 [M+H ]: calculated 390.1341, found 390.1330.

1 H NMR: (300 MHz, DMSO-d6): δ 13.69 (br s, 1H), 10.40 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 7.4 Hz, 2H), 7.67 (d, J = 7.2 Hz, 2H), 7.43 (d, J = 3.2 Hz, 1H), 7.41 (d, J = 7.4 Hz, 2H), 7.33 (dt, J = 7.4 Hz/1.0 Hz, 2H), 7.18 (dd, J = 9.1 Hz/3.0 Hz, 1H), 4.45 (d, J = 6.8 Hz, 2H), 4.33 (t, J = 6.8 Hz, 1H), 3.75 (s, 3H) ppm.

13 C NMR: (125 MHz, DMSO-d6): δ 169.2 (C), 153.8 (C), 152.9 (C), 143.7 (C), 140.8 (C), 134.0 (C), 127.7 (CH), 127.1 (CH), 125.0 (CH), 120.4 (CH), 120.2 (CH), 117.6 (C), 114.7 (CH), 66.1

(CH2), 55.4 (CH3), 46.5 (CH) ppm. IR (HATR): 3332 (vw), 2933 (vw), 1725 (s), 1673 (m), 1611 (w), 1591 (w), 1531 (m), 1476 (w), 1441 (w), 1424 (w), 1402 (w), 1315 (vw), 1279 (m), 1246 (s), 1208 (s), 1183 (s), 1154 (w), 1104

194

Part 3: Experimental Procedures

(w), 1090 (m), 1050 (m), 1032 (m) , 981 (w), 907 (w), 890 (w), 827 (w), 790 (w), 753 (m), 737 (s), 673 (m), 654 (w) cm-1.

TLC: Rf = 0.27 (Dichloromethane/MeOH 70/30).

XIII.2.5 N-(9-FLUORENYLMETHOXYCARBONYL)-2-AMINO-5-METHYLBENZOIC ACID VI.10

Recrystallization solvent: aqueous MeOH

Yield: 60 % (white powder), 4.5 g from 3 g 2-amino-5-

H methylbenzoic acid N O M.p.: 228 °C O Me COOH Molecular Formula: C23H19NO4

Molecular weight: 373.41 g mol. -1

LC-MS: Peak at 5.1 min; ES-MS negative mode [m/z (fragment, intensity)]: 372.1 (M-H+, 100).

1 H NMR: (500 MHz, DMSO-d6): δ 10.64 (s, 1H), 8.03 (br. d, J = 8.1 Hz, 1H), 7.92 (d, J = 7.5 Hz, 2H), 7.78 (d, J = 2.0 Hz, 1H), 7.69 (d, J = 7.5 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.39 (dd, J = 8.1 Hz/2.0 Hz, 1H), 7.35 (t, J = 7.4 Hz, 2H), 4.48 (d, J = 6.8 Hz, 2H), 4.35 (t, J = 6.8 Hz, 1H), 2.29 (s, 3H) ppm. 13 C NMR: (125 MHz, DMSO-d6): δ 169.6 (C), 152.6 (C), 143.6 (C), 140.7 (C), 138.4 (C), 134.7 (CH), 131.1 (CH), 131.0 (C), 127.6 (CH), 127.0 (CH), 124.9 (CH), 120.1 (CH), 118.4 (CH), 115.7

(C), 66.1 (CH2), 46.4 (CH), 20.0 (CH3) ppm. IR(HATR): 3318 (w), 2946 (w), 1729 (m), 1668 (s), 1590 (m), 1580 (m), 1524 (s), 1475 (m), 1448 (m),1415 (m), 1401 (m), 1316 (w), 1295 (w), 1252 (s), 1224 (s), 1207 (s), 1154 (m), 1100 (m), 1085 (vw), 1067 (m), 1036 (m), 962 (vw), 939 (vw), 899 (w), 932 (w), 791 (w), 757 (m), 738 (s), 697 (w), 670 w), 649 (w) cm-1.

TLC: Rf = 0.54 (Dichloromethane/MeOH 70/30).

195

Part 3: Experimental Procedures

XIII.2.6 N-(9-FLUORENYLMETHOXYCARBONYL)-2-AMINO-6-FLUOROBENZOIC ACID VI.11

Recrystallization solvent: EtOAc.

Yield: 55 % (beige powder), 4.0 g from 3 g 2-amino-6-fluorobenzoic acid. H N O M.P.: 181.5 °C O COOH F Molecular Formula: C22H16FNO4

Molecular weight: 377.36 g mol-1

LC-MS: Peak at 4.8 min; ES-MS negative mode [m/z (fragment, intensity)]: 376.0 (M-H+, 100), 179.9 (M-fluorenylmethanol-H+, 100).

1 H NMR: (300 MHz, DMSO-d6): δ 10.01 (s, 1H), 7.89 (d, J = 7.3 Hz, 1H), 7.89 (d, J = 7.3 Hz, 1H), 7.69 (d, J = 7.3 Hz, 2H), 7.20-7.60 (m, 6H), 7.01 (t, J = 9.5 Hz, 1H), 4.43 (d, J = 6.8 Hz, 2H), 4.31 (t, J = 6.6 Hz, 1H) ppm. 13 C NMR: (75 MHz, DMSO-d6): δ 166.1 (C), 160.7 (d, J = 252.5 Hz, C), 153.2 (C), 143.6 (C), 140.8 (C), 139.4 (d, J = 4.9 Hz, C), 133.0 (d, J = 11.0 Hz, CH), 127.7 (CH), 127.1 (CH), 125.1 (CH), 120.2 (CH), 117.2 (d, J = 2.7 Hz, CH), 111.7 (d, J = 15.4 Hz, C), 110.9 (d, J = 23.0 Hz,

CH), 66.3 (CH2), 46.5 (CH) ppm. IR (HATR): 3064 (w), 1700 (s), 1683 (s), 1611 (m), 1577 (m), 1527 (s), 1473 (m), 1463 (m), 1438 (m), 1398 (m), 1368 (m), 1326 (w), 1287 (m), 1256 (s), 1227 (s), 1211 (s), 1171 (m), 1113 (m), 1031 (w), 983 (m), 969 (m), 941 (w), 887 (vw), 831 (w), 809 (m), 794 (m), 780 (m), 768 (m), 757 (m), 739 (m), 731 (s), 718 (m), 689 (m), 649 (m) cm-1.

TLC: Rf = 0.30 (Dichloromethane/MeOH 70/30).

196

Part 3: Experimental Procedures

XIII.2.7 N-(9-FLUORENYLMETHOXYCARBONYL)-2-AMINO-4- TRIFLUOROMETHYLBENZOIC ACID VII.7

Recrystallization solvent: aqueous MeOH.

Yield: 62 % (white needles), 3.7 g from 3 g 2-amino-4-

H trifluoromethylbenzoic acid. F3C N O O M.P.: 218 °C COOH Molecular Formula: C23H16F3NO4

Molecular weight: 427.37 g mol-1.

LC-MS: Peak at 5.5 min; ES-MS negative mode [m/z (fragment, intensity)]: 426.1 (M-H+, 100).

HR-MS (ESI): Not detectable by mass spectrometer (positive and negative mode).

1 H NMR: (400 MHz, Acetone-d6): δ 8.84 (s, 1H), 8.31 (d, J=8.3 Hz, 1H), 7.89 (d, J=7.6 Hz, 2H), 7.73 (d, J =7.5 Hz, 2H), 7.49 -7.21 (m, 5H), 4.53 (d, J=7.1 Hz, 2H), 4.37 (t, J= 8.2 Hz, 1H) ppm.

13 C NMR: (100 MHz, Acetone-d6): major conformer: δ 169.8 (C), 154.4 (C), 145.2 (C), 142.6 (C), 136.2 (q, J= 32.6 Hz, C), 133.9 (CH), 129.1 (CH), 128.5 (CH), 128.4 (CH), 125.1 (q, J=272.3 Hz,

C), 126.5 (CH), 121.4 (CH), 119.1 (q, J=3.3 Hz, CH), 116.3 (q, J=3.8 Hz, CH), 68.4 (CH2), 48.2 (CH) ppm.

IR (HATR): 3204 (w), 2949 (w), 1713 (m), 1697 (m), 1622 (w), 1588 (m), 1534 (m), 1473 (m), 1436 (m), 1378 (m), 1329 (m), 1309 (m), 1297 (w), 1274 (m), 1251 (m), 1224 (m), 1197 (m), 1173 (s), 1135 (s), 1126 (s), 1098 (m), 1049 (s), 1000 (m), 921 (m), 891 (w), 844 (vw), 796 (m), 784 (m), 753 (m), 737 (s), 723 (s), 667 (m), 731 (m), 720 (vw) cm-1.

TLC: Rf = 0.16 (Dichloromethane/MeOH 70/30).

197

Part 3: Experimental Procedures

XIII.3 SYNTHESIS OF N-FMOC-3-AMINO-2-ALKYLPROPIONIC ACIDS

XIII.3.1 Synthesis of methyl 3-aryl-2-cyanoacrylates

XIII.3.1.1 General procedure

R 1 eq aldehyde, 0.1 eq AcOH NC COOMe 0.04 eq piperidine, dioxane, r.t, o.n NC COOMe

To a mixture of methyl cyanoacetate (124 mmol, 1 eq) and aldehyde (124 mmol, 1 eq) in dioxane (30 ml) is added acetic acid (12.4 mmol, 0.1 eq) and piperidine (5 mmol, 0.04 eq). The mixture is stirred overnight at room temperature. The reaction mixture was evaporated under reduced pressure, the residue was dissolved in 300 ml CH2Cl2 and washed with 300 ml water and 300 ml brine. The organic layer is concentrated under reduced pressure and the residue recrystallized from ethylacetate or purified by column chromatography (F.C.).

XIII.3.1.2 METHYL 3-(4-BROMOPHENYL)-2-CYANOACRYLATE VI.30

F.C.: Hexane/EtOAc 80/20 Br Yield: 87%

MeOOC CN Molecular Formula: C11H8BrNO2

Molecular weight: 266.09 g mol-1

LC-MS: Peak at 6.4 min, not detectable by mass spectrometer (positive and negative modes) HRMS (ESI): not detectable by mass spectrometer (positive and negative modes).

1 H NMR: (300 MHz, CDCl3): δ 8.21 (s, 1H), 7.87 (d, J = 8.7 Hz, 2H), 7.67 (d, J = 8.7 Hz, 2H), 3.96 (s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 162.8 (C), 153.8 (CH), 132.7 (CH), 132.3 (CH), 130.2 (C), 128.4

(C), 115.2 (C), 103.2 (C), 53.5 (CH3) ppm.

198

Part 3: Experimental Procedures

IR (HATR): 3099 (w), 3076 (w), 3032 (w), 2223 (m), 1720 (s), 1607 (s), 1580 (s), 1490 (m), 1427 (m), 1409 (m), 1370 (w), 1312 (w), 1272 (s), 1202 (s), 1009 (m), 986 (w), 967 (m), 840 (m), 821 (m), 792 (m), 762 (m), 706 (m) cm-1.

TLC: Rf = 0.31 (hexane/EtOAc 80/20).

XIII.3.1.3 METHYL 3-(4-CHLOROPHENYL)-2-CYANOACRYLATE VI.32

Yield: 85 % (White powder) Cl

Molecular Formula: C11H8ClNO2

MeOOC CN Molecular Weight: 221.6 g.mol-1

LC-MS: Peak at 6.4 min, not detectable by mass spectrometer (positive and negative modes)

HRMS (ESI): Not detectable by mass spectrometer (positive and negative mode).

1 H NMR: (500 MHz, CDCl3): δ 8.21 (s, 1H), 7.94 (d, J=8.5, 2H) 7.49 (d, J=8.5, 2H), 3.94 (s, 3H) ppm.

13 C NMR: (125 MHz, CDCl3): δ 162.8 (C), 153.7 (CH), 139.7 (C), 132.2 (CH), 129.8 (C), 129.7

(CH), 115.2 (C), 103.0 (C), 53.5 (CH3) ppm.

IR (HATR): 3079 (w), 3030 (w), 2960 (w), 1722 (s), 1606 (s), 1585 (s), 1562 (m), 1493 (m), 1452 (m), 1414 (m), 1370 (w), 1312 (m), 1271 (s), 1203 (s), 1112 (m), 1088 (s), 1048 (m), 1013 (m) , 966 (m), 837 (m), 817 (m), 794 (m), 761 (m), 709 (m), 670 (w) cm-1.

TLC: Rf = 0.33 (hexane/EtOAc 80/20).

XIII.3.1.4 METHYL 2-CYANO-3-(FURAN-2-YL)ACRYLATE VI.33

F.C.: hexane/Acetone 90/10

O Yield: 88% (orange solid)

MeOOC CN Molecular Formula: C9H7NO3

Molecular weight: 177.16 g mol-1

199

Part 3: Experimental Procedures

LC-MS: Peak at 5.3 min; ES-MS positive mode [m/z (fragment, intensity)]: 178.0 (M+H+, 100), 146.0 (M-MeO-, 35).

1 H NMR: (500 MHz, CDCl3): δ 8.04 (s, 1H), 7.77 (d, J =1.5 Hz, 1H), 7.41 (d, J =3.5 Hz, 1H), 6.68 (dd, J =3.7 Hz / 1.7 Hz, 1H), 3.92 (s, 3H) ppm.

13 C NMR: (125 MHz, CDCl3): δ 163.1 (C), 148.7 (C), 148.4 (CH), 139.7 (CH), 122.0 (CH), 115.3

(C), 114.0 (CH), 98.2 (C), 53.3 (CH3) ppm.

IR (HATR): 3128 (w), 3044 (m), 2958 (w), 2226 (m), 1913 (vw), 1726 (s), 1690 (vw), 1613 (s), 1540 (w), 1520 (vw), 1464 (w), 1450 (vw), 1428 (m), 1337 (vw), 1286 (w), 1250 (s), 1209 (s), 1154 (w), 1093 (s), 1066 (w), 1015 (s), 976 (m), 960 (w), 926 (w), 882 (w), 846 (w), 818 (w), 765 (vw), 752 (s), 705 (s), 674 (vw) cm-1.

TLC: Rf = 0.58 (dichloromethane/acetone 97/3).

XIII.3.1.5 METHYL 2-CYANO-3-(4-METHOXYPHENYL)ACRYLATE VI.34

Yield: 64% (white powder) OMe

Molecular Formula: C12H11NO3

MeOOC CN Molecular weight: 217.22 g mol-1

LC-MS: Peak at 6.0 min; ES-MS positive mode [m/z (fragment, intensity)]: 218.1 (M+H+, 100), 186.1 (M+MeO-, 25).

1 H NMR: (300 MHz, CDCl3): δ 8.18 (s, 1H), 8.01 (d, J=8.8 Hz, 2H), 7.00 (d, J=8.8 Hz, 2H), 3.92 (s, 3H), 3.90 (s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 163.9 (C), 163.6 (C), 154.6 (CH), 133.7 (CH), 124.3 (C), 116.2

(CN), 114.8 (CH), 98.8 (C), 55.6 (CH3), 53.2 (CH3) ppm.

IR (HATR): 3011 (w), 2961 (w), 2215 (m), 1723 (s), 1610 (w), 1583 (s), 1559 (s), 1514 (s), 1428 (m), 1263 (s), 1212 (s), 1182 (s), 1129 (m), 1089 (m), 1027 (m), 1012 (w), 970 (m), 948 (w), 833 (s), 806 (m), 757 (m), 747 (w), 726 (w), 627 (m) cm-1.

200

Part 3: Experimental Procedures

XIII.3.1.6 METHYL 3-(2-NAPHTHYL)-2-CYANOACRYLATE VI.35

Yield: 99 % (Yellow powder)

M.p.: 156.5 °C

MeOOC CN Molecular Formula: C15H11NO2

Molecular weight: 237.07 g mol-1

LC-MS: Peak at 6.6 min; ES-MS positive mode [m/z (fragment, intensity)]: 255.1 (M+NH4, 18), 238.0 (M+H+, 100)

+ + HRMS (ESI): C15H12NO2 [M+H ]: calculated 238.0868, found 238.0864.

1 H NMR: (300 MHz, CDCl3): δ 8.36 (d, J=7.7 Hz, 2H) 8.16 (d, J=8.7 Hz, 1H), 7.98-7.48 (m, 5H), 3.85 (s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 163.1 (C), 155.2 (CH), 135.4 (C), 134.2 (CH), 132.7 (C), 129.3 (CH), 129.1 (CH), 128.9 (C), 127.8 (CH), 127.2 (CH), 125.2 (CH), 125.1 (CH), 115.7 (C), 102.1

(C), 53.4 (CH3) ppm.

IR (HATR): 3026 (w), 2948 (w), 2921 (w), 2220 (s), 1725 (s, sharp), 1598 (s, sharp), 1571 (m), 1469 (w), 1457 (w), 1432 (m), 1378 (w), 1342 (w), 1277 (m), 1245 (s), 1220 (s), 1183 (m), 1161 (w), 1127 (w) , 1091 (m), 1022 (w), 979 (m), 963 (w), 911 (w), 894 (w), 869 (w), 860 (w), 813 (s), 759 (m), 748 (s), 719 (m), 643 (w) cm-1.

TLC: Rf = 0.20 (hexane/EtOAc 90/10).

XIII.3.1.7 METHYL 3-PHENYL-2-CYANOACRYLATE VI.36

F.C.: hexane/EtOAc 90/10

Yield: 91 % (white powder)

MeOOC CN Molecular Formula: C11H9NO2

Molecular weight: 187.19 g mol-1

LC-MS: Peak at 5.9 min; ES-MS positive mode [m/z (fragment, intensity)]: 205.1 (M+NH4, 35), 188.1 (M+H+, 100).

201

Part 3: Experimental Procedures

1 H NMR: (300 MHz, CDCl3): δ 8.26 (s, 1H, 8.05-7.95 (m, 2H), 7.63-7.45 (m, 2H), 3.94 (s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 162.9 (C), 155.3 (CH), 133.4 (CH), 131.3 (C), 131.1 (CH), 129.2

(CH), 115.4 (C), 102.5 (C), 53.3 (CH3) ppm.

TLC: Rf = 0.21 (hexane/EtOAc 90/10).

XIII.3.2 Synthesis of 3-substituted methyl 2-cyanopropionates

XIII.3.2.1 Synthesis of methyl 2-cyano-4-methylpentanoate VI.42

1 eq isobutyraldehyde, 0.1 eq AcOH NC COOMe 0.04 eq piperidine, dioxane, NC COOMe VI.46 Pd/C 5wt%, 2h, r.t VI.42

To a mixture of methyl cyanoacetate (15 g, 150.5 mmol) and isobuteraldehyde (13.6 g, 150 mmol) in dioxane (45 ml) is added acetic acid (870 µl, 15 mmol), piperidine (600 µl, 15 mmol) and (300 mg, Pd/C 10 wt % (300 mg).The mixture is stirred under a hydrogen atmosphere (2 barr) at room temperature for 2 hours; then filtered on celite and the filterate concentrated under reduced pressure. The residue is dissolved in 60 ml ethyl acetate and washed with water and brine. The organic layer is concentrated under reduced pressure and the residue is purified by flash chromatography (F.C.) (Silica gel, Hexane/ EtOAc, 95/5).

Yield: 82% (Colorless liquid)

Molecular Formula: C8H13NO2

NC COOMe Molecular weight: 155.19 g.mol-1

LC-MS: Peak at 5.7 min; ES-MS negative mode [m/z (fragment, intensity)]: 154.2 (M-H+, 90).

- + HRMS (ESI): C8H12NO2 [M-H ]: calculated 154.0868, found 154.0880.

1 H NMR: (500 MHz, CDCl3): δ 3.80 (s, 3H), 3.53 (dd, J= 5.8/5.0Hz, 1H), 1.74-1.90 (m, 3H), 0.97 (dd, J= 6.2/6.3 Hz, 6H) cm-1.

202

Part 3: Experimental Procedures

13 C NMR: (125 MHz, CDCl3): δ 166.9 (C), 116.5 (C), 53.4 (CH3), 38.4 (CH2), 35.7 (CH), 26.1

(CH), 22.3 (CH3), 21.2 (CH3) ppm.

IR (HATR): 3390 (w), 2959 (w), 2250 (vw), 1746.4 (s, sharp), 1469 (w), 1436 (m), 1389 (w), 1371 (w), 1335 (w), 1308 (w), 1268 (m), 1248 (m), 1219 (m), 1200 (m), 1172 (m), 1135 (w), 1114 (w), 1008 (m), 960 (vw), 935 (vw), 910 (vw), 832.7 (vw), 763 (vw), 641 (vw), 617 (vw) cm-1.

TLC: Rf = 0.16 (Hexane/EtOAc 95/5).

XIII.3.2.2 Synthesis of methyl 3-aryl-2-cyanopropionates

XIII.3.2.2.1 General procedure

R R AcOH 0.4 M, 1.1 eq NaBH3CN NC COOMe MeOH/CH2Cl2 (5/1), 40 min, r.t NC COOMe

To a solution of the acrylate (22.3 mmol, 1 eq) in MeOH/ CH2Cl2 (5/1, 300 ml) is added NaBH3CN (25 mmol, 1 eq), and a small amount of p-bromocresol “green”. The mixture is acidified with AcOH (50 mmol, 2 eq), turning the color to yellow. The reaction is stirred overnight at room temperature, after which water is added and the product extracted using CH2Cl2 (3x 250 ml). The combined organic layers are concentrated under reduced pressure, the residue is purified by flash chromatography (F.C.)

XIII.3.2.2.2 METHYL 3-(4-CHLOROPHENYL)-2-CYANOPROPIONATE VI.37

F.C.: hexane/EtOAc 90/10 Cl Yield: 89% (yellow oil)

NC COOMe Molecular Formula: C11H10ClNO2

Molecular Weight: 223.6 g mol-1

LC-MS: Peak at 6.0 min; ES-MS negative mode [m/z (fragment, intensity)]: 224.0 (M(37Cl)-H+, 33), 222.0 (M(35Cl)-H+, 100).

203

Part 3: Experimental Procedures

35 - + HRMS (ESI): C11H9 ClO2 [M-H ]: calculated 222.0322, found: 222.0332.

1 H NMR: (300 MHz, CDCl3): δ 7.32 (dt, J=8.7/2.5 Hz, 2H), 7.20 (dt, J=8.7/2.3 Hz, 2H), 3.81 (s, 3H), 3.73 (dd, J = 8.1 Hz / 5.8 Hz, 1H), 3.25 (dd, J=13.9/6.2 Hz, 1H), 3.18 (dd, J=13.9/8.4 Hz, 1H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 165.7 (C), 133.8 (C), 133.6 (C), 130.4 (CH), 129.0 (CH), 115.8

(CN), 53.6 (CH3), 39.3 (CH), 35.0 (CH2) ppm.

IR (HATR): 3078 (w), 3029 (w), 2959 (w), 2919 (w), 2846 (vw), 2223 (m), 1723 (s, sharp), 1606 (m), 1585 (m), 1562 (m), 1493 (m), 1413 (m), 1370 (w), 1312 (w), 1272 (s), 1203 (s), 1191 (s), 1112 (m), 1088 (s), 1013 (w), 967 (m), 836 (m), 819 (m), 794 (m), 761 (m), 709 (w), 670 (w), 626 (w) cm-1.

TLC: Rf =0.23 (hexane/ EtOAc 80/20).

XIII.3.2.2.3 METHYL 3-(4-BROMOPHENYL)-2-CYANOPROPANOATE VI.27

F.C.: Hexane/EtOAc 80/20 Br Yield: 81% (Colorless oil)

NC COOMe Molecular Formula: C11H10BrNO2

Molecular weight: 268.11 g mol-1

LC-MS: Peak at 6.1 min; ES-MS negative mode: [m/z (fragment, intensity)] 268.0 (M(81Br) - H+,100), 266.0 (M(79Br) -H+,100).

79 - + HRMS (ESI): C11H9 BrNO2 [M-H ]: calculated 265.9817, found 265.9829.

1 H NMR: (300 MHz, CDCl3): δ 7.44-7.37 (m, 2H), 7.12-7.05 (m, 2H), 3.73 (s, 3H), 3.65 (dd, J= 8.1/5.8 Hz, 1H), 3.22-3.04 (m, 2H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 165.7 (C), 134.1 (C), 132.1 (CH), 130.8 (CH), 122.0 (C), 115.7

(CN), 53.7 (CH3), 39.2 (CH), 35.0 (CH2) ppm.

204

Part 3: Experimental Procedures

IR(HATR): 3026 (w), 2954 (w), 2251 (w), 1744 (s), 1593 (w), 1488 (m), 1436 (m), 1406 (w), 1345 (w), 1264 (m), 1212 (m), 1180 (w), 1162 (w), 1105 (w), 1072 (m), 1012 (m), 963 (vw), 916 (vw), 894 (w), 845 (vw), 826 (w), 803 (m), 781 (w), 714 (m), 642 (vw), 619 (w) cm-1.

TLC: Rf = 0.19 (hexane/EtOAc 80/20).

XIII.3.2.2.4 METHYL 2-CYANO-3-(FUR-2-YL)PROPANOATE VI.38

Yield: 96%, (brown oil)

O Molecular Formula: C9H9NO3 NC COOMe Molecular weight: 179.17 g mol-1

LC-MS: Peak at 5.2 min; ES-MS negative mode [m/z (fragment, intensity)]: 178.0 (M-H+,100), 118.1 (12).

1 H NMR: (300 MHz, CDCl3): δ 7.39-7.36 (m, 1H), 6.36-6.32 (m, 1H), 6.28-6.25 (m, 1H), 3.89- 3.82 (m, 1H), 3.83 (s, 3H), 3.40-3.23 (m, 2H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 165.6 (C), 148.8 (C), 142.6 (CH), 115.7 (C), 110.6 (CH), 108.4

(CH), 53.7 (CH3), 36.9 (CH), 28.5 (CH2) ppm.

IR(HATR): 3128 (w), 3044 (m), 2958 (w), 2226 (m), 1726 (s), 1690 (vw), 1613 (s), 1540 (m), 1520 (vw), 1464 (w), 1450 (vw), 1428 (m), 1337 (w), 1286 (w), 1250 (s), 1209 (s), 1154 (w), 1093 (s), 1066 (w), 1015 (s), 976 (w), 160 (w), 926 (m), 882 (m), 846 (w), 818 (m), 752 (s), 705 (s), 674 (vw) cm-1.

TLC: Rf = 0.20 (hexane/EtOAc 80/20).

205

Part 3: Experimental Procedures

XIII.3.2.2.5 METHYL 2-CYANO-3-(4-METHOXYPHENYL)PROPIONATE VI.39

Molecular Formula: C12H13NO3 OMe Molecular Weight: 219.23 g mol-1

1 NC COOMe H NMR: (300 MHz, CDCl3): δ 7.18-7.13 (m, 2H), 6.88-6.81 (m, 2H), 3.74 (s, 3H), 3.73 (s, 3H), 3.70 (dd, J = 8.1/5.8 Hz, 1H), 3.21-3.06 (m, 2H).

TLC: Rf = 0.15 (hexane/EtOAc 80/20).

XIII.3.2.2.6 METHYL 2-CYANO-3-(NAPHTH-2-YL)PROPIONATE VI.40

F.C.: Hexane/ EtOAc 90/10

Yield 97% (White powder)

NC COOMe M.p. 82 °C

Molecular Formula: C15H13NO2

Molecular Weight: 239.26 g mol-1

LC-MS: Peak at 6.2 min, ES-MS negative mode [m/z (fragment, intensity)]: 238.1 (M-1, 100), 239.0 (M, 18).

- + HRMS (ESI): C15H12NO2 [M-H ]: calculated 238.0946, found: 238.0883.

1 H NMR: (300 MHz, CDCl3): δ 7.88-7.79 (m, 3H), 7.76 (br.s, 1H), 7.55-7.43 (m, 2H), 7.38 (dd, J= 8.5/1.9 Hz, 1H), 3.85 (dd, J= 8.3/5.7 Hz, 1H), 3.80 (s, 3H), 3.49 (dd, J= 13.8/5.7, 1H), 3.38 (dd, J= 13.8/8.5 Hz, 1H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 166.0 (C), 133.4 (C), 132.7 (C), 132.6 (C), 128.7 (CH), 128.1 (CH), 127.8 (CH), 127.7 (CH), 126.6 (CH), 126.4 (CH), 126.2 (CH), 116.03 (CN), 53.6 (CH), 39.5

(CH3), 35.9 (CH2) ppm.

IR(HATR): 3053 (w), 3023 (w), 2969 (w), 2925 (w), 2251 (w), 1757 (s), 1738 (s, sharp), 1631 (w), 1599 (w), 1508 (w), 1429 (m), 1367 (w), 1322 (m), 1267 (s), 1251 (s), 1227 (s), 1213 (s), 1185 (m), 1147 (m), 1164 (m), 1123 (m), 1072 (m), 1028 (m), 1015 (m), 979 (m), 951 (m), 903

206

Part 3: Experimental Procedures

(m), 882 (w), 863.8 (m), 839 (w), 814 (s, sharp), 791 (w), 740 (s), 716 (w), 655 (m), 624 (w) cm- 1.

TLC: Rf = 0.24 (hexane/EtOAc 80/20).

XIII.3.2.2.7 METHYL 2-CYANO-3-PHENYLPROPIONATE VI.41

F.C.: Hexane/EtOAc 90/10

Yield: 89% (colorless liquid) NC COOMe

Molecular Formula: C11H11NO2

Molecular Weight: 189.21 g mol-1

LC-MS: Peak at 5.7 min; ES-MS negative mode [m/z (fragment, intensity)]: 188.1 (M-1, 100).

1 H NMR: (300 MHz, CDCl3): δ 7.39-7.35 (m, 1H), 7.34-7.31 (m, 2H), 7.30-7.25 (m, 2H), 3.80 (s, 3H), 3.78-3.73 (m, 1H), 3.33-3.16 (m, 2H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 166.0 (C), 135.2 (C), 129.1 (CH), 128.9 (CH), 128.7 (CH), 127.8

(CH), 127.6 (CH), 116.1 (CN), 53.5 (CH3), 39.5 (CH), 35.7 (CH2) ppm.

TLC: Rf = 0.24 (hexane/EtOAc 90/10).

XIII.3.3 Synthesis of methyl 3-(tert-butoxycarbonyl)amino-2-alkylpropionates

XIII.3.3.1 General procedure

R R 0.1 eq NiCl2.6H2O, 7 eq NaBH4 Boc HN NC COOMe 2 eq Boc2O, 0.2 M MeOH, 0 to r.t COOMe

To a cooled (0 °C) solution of the nitrile (122 mmol, 1 eq) in MeOH (610 ml) is added Boc2O (244 mmol, 2 eq) and NiCl2.6H2O (12 mmol, 0.1 eq). Afterwards NaBH4 (855 mmol, 7 eq) is added protionwise (0.22 eq every 10 min). After the addition, the reaction is stirred overnight under N2

207

Part 3: Experimental Procedures gas. The reaction mixture was concentrated under reduced pressure and the residue is dissolved in ethylacetate. After washing with a saturated aqeous solution of NaHCO3, the organic layer is dried over magnesium sulfate. After filtering the drying agent, the solvent is removed under reduced pressure and the residue is purified by flash chromatography (F.C.).

XIII.3.3.2 METHYL 3-(TERT-BUTOXYCARBONYL)AMINO-2-(4-BROMOBENZYL)- PROPIONATE VI.44

F.C.: Hexane/EtOAc 80/20 Br

O O Yield: 49% (colourless oil)

HN COOMe Molecular Formula: C16H22BrNO4

Molecular weight: 372.25 g mol-1

LC-MS: Peak at 6.8 min; ES-MS positive mode: [m/z (fragment, intensity)]: 318.0 (M(81Br)- isobutene+H+, 5), 316.0 (M(79Br)-isobutene+H+, 5), 286.0 (6), 284.0 (6), 274.0 (M(81Br)- + 79 + isobutene-CO2+2H , 100), 272.0 (M( Br)-isobutene-CO2+2H , 100), 242.0 (10), 240.0 (10).

79 + + HRMS (ESI): C11H15 BrNO2 [M- isobutene-CO2+2H ]: calculated: 272.0286, found 272.0281.

1 H NMR: (500 MHz, CDCl3): δ 7.33 (d, J = 8.2 Hz, 2H), 6.97 (d, J = 8.2 Hz, 2H), 4.73 (br.s, NH), 3.57 (s, 3H), 3.40-3.10 (m, 2H), 2.90-2.76 (m, 2H), 2.75-2.60 (m, 1H), 1.36 (s, 9H) ppm.

13 C NMR: (125 MHz, CDCl3): δ 174.4 (C), 155.8 (C), 137.3 (C), 131.6 (CH), 130.6 (CH), 120.5

(C), 79.5 (C), 51.9 (CH3), 47.2 (CH), 41.5 (CH2), 35.2 (CH2), 28.3 (CH3) ppm.

IR(HATR): 3285 (vw), 2976 (vw), 2951 (vw), 1730 (m), 1690 (w), 1621 (s), 1530 (w), 1483 (w), 1417 (m), 1366 (m), 1249 (w), 1206 (vw), 1163 (m), 1072 (vw), 1012 (m), 954 (vw), 849 (w), 815 (vw) cm-1.

TLC: Rf = 0.11 (hexane/EtOAc 90/10).

208

Part 3: Experimental Procedures

XIII.3.3.3 METHYL 3-(TERT-BUTOXYCARBONYL)AMINO-2-(4-CHLOROBENZYL)- PROPIONATE VI.46

F.C.: Hexane/Acetone 95/5 Cl

O O Yield: 58 % White powder

HN COOMe Molecular Formula: C16H22ClNO4

Molecular Weight: 327.8 g mol-1

LC-MS: Peak at 6.651 min, ES-MS positive mode [m/z (fragment, intensity)]: 240.1 (6), 230.1 37 + 35 + 35 (M( Cl)-isobutene-CO2+H , 33), 228.1 (M( Cl)-isobutene-CO2+H , 100), 198 (M( Cl)- - 37 - isobutene-CO2-OMe , 12), 196 (M( Cl)-isobutene-CO2-OMe , 5).

35 + + HRMS (ESI): Peak for C11H15 ClNO2 [M-isobutene-CO2+2H ]: calculated 228.0791, found: 228.086.

1 HNMR: (300 MHz, CDCl3): δ 7.36-7.21 (m, 2H), 7.13 (dt, J= 8.3/2.5 Hz, 2H), 4.89 (br.s, NH), 3.64 (s, 3H), 3.42-2.74 (m, 5H), 1.42 (s, 9H) ppm.

13 CNMR: (75 MHz, CDCl3): δ 174.3 (C), 155.7 (C), 136.7 (C), 132.4 (C), 130.1 (CH), 128.6 (CH),

128.5 (CH), 126.5 (CH), 79.4 (C), 51.8 (CH3), 47.2 (CH), 41.4 (CH2), 35.1 (CH2), 28.2 (CH3) ppm.

IR (HATR): 3405 (vw), 3079 (w), 3030 (w), 2960 (w), 1721 (s), 1606 (m), 1585 (s), 1563 (m), 1493 (m), 1425 (m), 1414 (m), 1370 (w), 1312 (m), 1270 (s), 1203 (s), 1112 (m), 1087 (s), 1013 (m), 966 (m), 903 (w), 837 (s), 817 (m), 784 (m), 761 (m), 709 (m), 670 (w), 626 (w) cm-1.

TLC: Rf = 0.11(hexane/ EtOAc 90/10).

209

Part 3: Experimental Procedures

XIII.3.3.4 METHYL 3-(TERT-BUTOXYCARBONYL)AMINO-2-(FUR-2- YLMETHYL)PROPIONATE VI.47

F.C.: hexane/EtOAc 90/10 to 60/40 O O O Yield: 40% (brown oil) HN COOMe Molecular Formula: C14H21NO5

Molecular weight: 283.32 g mol-1

LC-MS: Peak at 6.0 min; ES-MS positive mode [m/z (fragment, intensity)]: 184.1 (M-isobutene- + CO2+2H , 100), 166.1 (7), 152.0 (18), 123.0 (7).

1 H NMR: (300 MHz, CDCl3): δ 7.26-7.15 (m, 1H), 6.22-6.17(m, 1H), 5.99 (d, J= 3.4 Hz, 1H), 4.82 (br s, 1H), 3.62 (s, 3H), 3.40-3.10 (m, 2H), 3.00-2.70 (m, 3H), 1.36 (s, 9H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 174.2 (C), 155.8 (C), 152.1 (C), 141.6 (CH), 110.3 (CH), 106.8

(CH), 79.4 (C), 52.0 (CH3), 44.7 (CH), 41.2 (CH2), 28.3 (CH3), 28.0 (CH2) ppm.

IR(HATR): 3357 (br.m), 2976 (w), 2930 (w), 1706 (s), 1506 (s), 1467 (m), 1390 (w), 13650 (m), 1161 (s), 1097 (w), 1076 (w), 1047 (w), 1008 (m), 971 (w), 928 (m), 885 (w), 857 (w), 836 (w), 804 (w), 782 (w), 733 (m) cm-1.

TLC: Rf = 0.79 (dichloromethane/MeOH 90/10).

XIII.3.3.5 METHYL 3-(TERT-BUTOXYCARBONYL)AMINO-2-(NAPHTH-2-YLMETHYL) PROPANOATE VI.48

F.C.: Hexane/EtOAc 90/10

O O Yield: 57% (White powder)

HN COOMe M.p.: 92.5 °C

Molecular Formula: C20H25NO4

Molecular Weight: 343.41 g mol-1

210

Part 3: Experimental Procedures

LC-MS: Peak at 6.8 min; ES-MS positive mode [m/z (fragment, intensity)]: 329.2 (10), 288.2 (M- + + isobutene+H , 100), 244.2 (M-isobutene-CO2+H , 95).

+ + HRMS (ESI): C20H25NO4Na [M+Na ]: calculated 366.1681, Found: 366.1667.

1 H NMR: (500 MHz, CDCl3): δ 7.82-7.77 (m, 3H), 7.62 (s, 1H), 7.48-7.42 (m, 2H), 7.32-7.30 (dd, J=8.4/1.6 Hz, 1H), 4.90 (br.s, NH), 3.65 (s, 3H), 3.45-3.30 (m, 2H), 3.15-3.11(m, 1H), 3.07-2.97 (m, 2H), 1.44 (s, 9H) ppm.

13 C NMR: (125 MHz, CDCl3): δ 174.6 (C), 155.8 (C), 135.8 (C), 133.5 (C), 132.3 (C), 128.2 (CH),

127.6 (CH), 127.6 (CH), 127.4 (CH), 127.1(CH), 126.1 (CH), 125.5(CH), 79.5 (C), 51.9 (CH3),

47.4 (CH), 41.7 (CH2), 36.1 (CH2), 28.4 (CH3) ppm.

IR (HATR) : 3382 (w), 3052 (w), 2975 (w), 2926 (w), 1711 (s), 1632 (w), 1600 (w), 1507 (m), 1436 (m), 1390 (w), 1365 (w), 1249 (m), 1199 (m), 1162 (s), 1094 (m), 1051 (m), 1018 (m), 975 (w), 895 (w), 856 (w), 816 (m), 781 (w), 747 (m), 650 (w) cm-1.

TLC: Rf = 0.12 (hexane/EtOAc 90/10).

XIII.3.3.5 METHYL 3-(TERT-BUTOXYCARBONYL)AMINO-2-ISOBUTYL-PROPIONATE VI.49

F.C.: (Pentane/EtOAc 96/4 to 90/10) O O Yield: 74% (Colorless liquid) HN COOMe Molecular Formula: C13H25NO4

Molecular weight: 259.34 g.mol-1

LC-MS Peak at 6.4 min; ES-MS positive mode [m/z (fragment, intensity)]: 172.1 (25), 160.1 (M- + isobutene-OMe+H , 100), 128.1 (M-isobutene-CO2-OMe, 34), 99.1 (M-isobutene-CO2- COOMe+H+, 18).

HRMS (ESI): not detectable by mass spectrometer (positive and negative modes).

1 H NMR (300 MHz, CDCl3): δ 4.85 (br.s, NH), 3.69 (s, 3H), 3.38-3.17 (m, 2H), 2.73-2.64 (m, 1H), 1.68-1.50 (m, 2H), 1.43 (s, 9H), 1.35-1.22 (m, 1H), 0.90 (d, J =6.4 Hz, 6H) ppm.

211

Part 3: Experimental Procedures

13 C NMR (125 MHz, CDCl3): δ 175.8 (C), 155.8 (C), 79.3 (C), 51.7 (CH3), 43.7 (CH), 41.9 (CH2),

38.7 (CH2), 28.3 (3 CH3), 25.85 (CH), 22.53 (CH3), 22.38 (CH3) ppm.

IR (HATR) 3377 (w), 2955 (m), 2930 (m), 2870 (w), 1715 (s), 1700 (s), 1509 (m), 1466 (w), 1453 (w), 1432 (w), 1389 (w), 1365 (m), 1338 (w), 1268 (m), 1247 (m), 1149 (m), 1164 (s), 1072 (w), 1041 (w), 1025 (w), 967 (w), 768 (w), 759 (vw), 861 (w), 833 (w), 780 (w), 775 (w), 730 (vw), 725 (vw), 680 (w), 650 (w), 614 (w), 601 (w) cm-1.

TLC Rf = 0.16 (hexane/EtOAc 95/5).

XIII.3.4 Synthesis of 3-(tert-butoxycarbonyl)amino-2-alkylpropionic acids

XIII.3.4.1 General procedure

R R Boc A: 2 eq NaOH, H2O/MeOH (2/1), 50°C, 20 min Boc HN HN COOMe B: 2 eq NaOH, H2O/MeOH/dioxane (2/1/1),r.t, o.n COOH

A: The methyl ester (23 mmol, 1 eq) is dissolved in a mixture of H2O and MeOH (2/1) (500 ml) then NaOH (69.4 mmol, 3 eq) is added. The reaction is refluxed at 50° C for 20 min. The reaction mixture is concentrated under reduced pressure, after which the residue is purified by column chromatography.

B: To a solution of methyl ester (20.53 mmol, 1 eq) in H2O/MeOH/dioxane (2/1/1) (100 ml), is added NaOH (41.06 mmol, 2 eq). The reaction mixture is stirred overnight at room temperature, after which it is acidified with a 1 M aqueous KHSO4 solution to pH 2. The product is extracted with ethyl acetate (600 ml) and filtered over silica.

212

Part 3: Experimental Procedures

XIII.3.4.2 2-(4-BROMOBENZYL)-3(TERT-BUTOXYCARBONYL)AMINOPROPIONIC ACID VI.50

Procedure: B Br F.C.: hexane/EtOAc/AcOH 90/10/1 O O HN Yield: 65% (white powder) COOH

Molecular Formula: C15H20BrNO4

Molecular weight: 358.23 g mol-1

LC-MS: Peak at 4.6 min; ES-MS negative mode: [m/z (fragment, intensity)]: 358.0 (M(81Br)-H+, 100), 356.0 (M(79Br)-H+, 100), 283.9 (M(81Br)-tBuOH-H+, 40), 281.9 (M(79Br)-tBuOH-H+, 40).

79 + + HRMS (ESI): C10H13 BrNO2 [M-isobutene-CO2+2H ]: calculated 258.0130, found 258.0123.

1 H NMR: (300 MHz, CDCl3): δ 7.34 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H), 6.46 (2x br.s, NH), 3.4-2.5 (m, 5H), 1.37 (s, 9H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 178.7 (C), 157.9 (C), 137.0 (C), 134.1 (C), 131.7 (CH), 130.6

(CH), 74.3 (C), 47.4 (CH), 42.1 (CH2), 35.0 (CH2), 28.3 (CH3) ppm.

TLC: Rf = 0.08 (hexane/EtOAc/AcOH 80/20/1).

XIII.3.4.3 3-(TERT-BUTOXYCARBONYL)AMINO-2-(4-CHLOROBENZYL)PROPIONIC ACID VI.51

Cl Procedure: B

O O F.C.: hexane/EtOAc/AcOH 70/30/1 HN COOH Yield: 99% (White powder)

Molecular Formula: C15H20ClNO4

Molecular Weight: 313.77 g mol-1

213

Part 3: Experimental Procedures

LC-MS: Peak at 4.5 min; ES-MS negative mode [m/z (fragment, intensity)]: 627.1 (M(37Cl)+M(35Cl)-H+, 20), 625.1 (M(35Cl)+M(35Cl)-H+, 25), 314.1 (M(37Cl)-H+, 31), 313.1 (20), 312.0 (M(35Cl)-H+, 100), 238.0 (M(35Cl)-tBuOH-H+,40), 239.0 (5), 240.0 (M(37Cl)-t-BuOH-H+, 10).

35 - + HRMS (ESI): C15H19 ClNO4 [M-H ]: calculated 312.1003, found: 312.1007.

1 H NMR: (300 MHz, CDCl3): δ 7.32-7.18 (m, 2H), 7.16-7.08 (m, 2H), 6.53 and 4.97 (2x br.s, NH), 3.48-2.60 (m, 5H), 1.42 (s, 9H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 178.6 (C), 155.9 (C), 136.5 (C), 132.5 (C), 130.2 (CH), 128.1

(CH), 79.8 (C), 47.1 (CH), 41.2 (CH2), 34.9 (CH2), 28.3 (CH3) ppm.

IR(HATR): 3317 (m), 3070 (w), 2983 (m), 2933 (w), 2899 (w), 1703 (s, sharp), 1637 (s), 1560 (w), 1493 (m), 1478 (m), 1462 (m), 1452 (m), 1435 (m), 1409 (s), 1364 (m), 1340 (m), 1296 (m), 1283 (s), 1256 (m), 1228 (m), 1202 (m), 1160 (m), 1141 (m), 1089 (m), 1068 (m), 1026 (m), 1013 (m), 969 (m), 969 (w), 853 (w), 842 (m), 812 (w), 793 (m), 773 (m), 701 (m), 679 (m) cm-1.

TLC: Rf =0.17 (hexane/EtOAc/AcOH 70/30/1).

XIII.3.4.4 3-(TERT-BUTOXYCARBONYL)AMINO-2-(FUR-2-YLMETHYL)PROPIONIC ACID VI.52

Procedure: B

O O O Yield: 78% (yellow powder) HN COOH Molecular Formula: C13H19NO5

Molecular weight: 269.29 g mol-1

LC-MS: Peak at 4.0 min; ES-MS positive mode: [m/z (fragment, intensity)]: 268.1 (M-H+, 93), 194.0 (M-tBuOH-H+, 100).

- + HRMS (ESI): Peak for C13H18NO5 [M-H ]: calculated 268.1185, found 268.1188.

1 HNMR: (300 MHz, CDCl3): δ 7.31-7.29 (m, 1H), 6.46 and 4.99 (2x br.s, NH), 6.27-6.26 (m, 1H), 6.05-6.11 (m, 1H), 3.48-2.71 (m, 5H), 1.42 (s, 9H) ppm.

214

Part 3: Experimental Procedures

13 CNMR: (75 MHz, CDCl3): δ 178.5 (C), 156.0 (C), 152.0 (C), 141.6 (CH), 110.3 (CH), 107.0

(CH), 81.3 (C), 45.0 (CH), 42.2 (CH2), 28.3 (CH3), 27.9 (CH2) ppm.

IR(HATR): 3380 (m), 2982 (w), 2940 (w), 1681 (s), 1599 (w), 1520 (s), 1437 (m), 1391 (m), 1367 (m), 1327 (w), 1270 (w), 1249 (s), 1195 (vw), 1181 (vw), 1157 (s), 1081 (w), 1056 (m), 1010 (m), 939 (s), 899 (m), 884 (w), 856 (w), 839 (w), 798 (w), 783 (w), 734 (s), 676 (w) cm-1.

TLC: Rf = 0.09 (hexane/EtOAc/AcOH 80/20/1).

XIII.3.4.5 3-(TERT-BUTOXYCARBONYL)AMINO-2-(NAPHTH-2-YLMETHYL) PROPIONIC ACID VI.53

Procedure: A

O O F.C.: hexane/EtOAc/AcOH 85/15/10 to 80/20/10 HN COOH Yield 98% (White powder)

M.P. 124 °C

Molecular Formula: C19H23NO4

Molecular Weight: 329.39 g mol-1

LC-MS: Peak at 4.6 min; ES-MS negative mode [m/z (fragment, intensity)]: 328.1 (M-H+, 100).

- + HRMS (ESI): C15H12NO2 [M-H ]: calculated 328.1548, Found: 328.1552.

1 H NMR: (500 MHz, Acetone-d6): δ 7.86-7.82 (m, 3H), 7.75 (s, 1H), 7.49-7.42 (m, 3H), 6.08 (br.s, NH), 3.36-3.35 (m, 2H), 3.12-3.01 (m, 3H), 1.40 (s, 9H) ppm.

13 C NMR: (125 MHz, Acetone-d6): δ 175.3 (C), 138.0 (C), 134.7 (C), 133.4 (C), 128.8 (CH), 128.5

(CH), 128.5 (CH), 128.3 (CH), 126.9 (CH), 126.3 (CH), 78.8 (C), 48.2 (CH), 42.9 (CH2), 36.6

(CH2), 28.6 (CH3) ppm.

IR (HATR): 3399 (m), 3054 (w), 2976 (m), 2942 (w), 2897 (w), 1688 (s, sharp), 1601 (w), 1514 (m), 1443 (m), 1392 (m), 1365 (m), 1309 (w), 1271 (m), 1256 (m), 1241 (m), 1168 (s), 1097 (w), 1055 (w), 963 (w), 909 (w), 896 (w), 876 (w), 862 (w), 813 (m), 771 (w), 751 (m), 657 (w) cm-1.

215

Part 3: Experimental Procedures

TLC: Rf = 0.11 (hexane/EtOAc/AcOH 85/15/1).

XIII.3.4.6 2-(TERT-BUTOXYCARBONYL)AMINOMETHYL-4-METHYLPENTANOIC ACID VI.54

Procedure: A

O O F.C.: hexane/EtOAc/AcOH 70/30/1 HN COOH Yield: 97% (White powder)

M.p.: 114 °C

Molecular Formula: C12H23NO4

Molecular weight: 245.31

LC-MS: Peak at 4.8 min; ES-MS negative mode [m/z (fragment, intensity)]: 170.1 (M-tBuOH-H+, 90), 244.1 (M-H+, 100), 245.1 (M, 18).

- + HRMS (ESI): C12H22NO4 [M-H ] calculated: 244.1548, found: 244.1557.

1 H NMR (500 MHz, Acetone-d6): δ 5.97 (br.s, 1H), 3.15-3.28 (m, 2H), 2.78-2.65 (m, 1H), 1.68- 1.66 (m, 1H), 1.53-1.50 (m, 1H), 1.39 (s, 9H), 1.33-1.29 (m, 1H), 0.91 (d, J=6.3 Hz, 6H) ppm.

13 C NMR (125 MHz, Acetone-d6): δ 156.7 (C), 78.8 (C), 44.1 (CH), 43.4 (CH2), 39.7 (CH2), 28.7

(CH3), 26.8 (CH), 23.3 (CH3), 22.6 (CH3) ppm.

IR (HATR): 3377 (w), 2956 (m), 2932 (m), 2871 (m), 1710 (s), 1650 (vw), 1510 (m), 1455 (m), 1391 (m), 1336 (m), 1351 (m), 1270 (m), 1251 (m), 1168 (s), 1120 (s), 1102 (s), 1068 (s), 984 (vw), 967 (w), 934 (w), 915 (vw), 889 (w), 865 (m), 843 (vw), 807 (w), 776 (vw), 743 (vw), 635 (vw), 620 (vw) cm-1.

TLC: Rf = 0.36 (hexane/EtOAc/AcOH 70/30/1).

216

Part 3: Experimental Procedures

XIII.3.5 Synthesis of HCl salts of 3-amino-2-alkylpropionic acids

XIII.3.5.1 General procedure

R R Boc Dioxane/conc. HCl 9/1 HN HCl.H2N COOH r.t , 7 h COOH

To a solution of Boc-amino acid (13.5 mmol, 1 eq), in dioxane (120 ml) is added 13.5 ml concentrated HCl and the obtained colorless solution is stirred for 7 h at room temperature. The reaction mixture is concentrated under reduced pressure to give the product as a white solid. The products are used as such in the next step.

XIII.3.6 Synthesis of 3-(9-Fluorenylmethyloxycarbonyl)amino-2-alkylpropionic acids

XIII.3.6.1 General procedure

R R Fmoc 1 eq FmocOSu, 1 eq NaHCO3 HCl.H2N HN COOH THF/H2O 2/1 COOH

To a solution of crude amino acid hydrochloride (8.9 mmol, 1 eq) in water (30 ml), is added sodium bicarbonate (8.93 mmol, 1 eq). After 5 minutes THF (60) ml and Fmoc-OSu (8.93 mmol, 1 eq) are added and the reaction mixture is stirred overnight at room temperature. The resulting white suspension is acidified using 1 M HCl to pH 1 and the resulting clear solution is extracted with EtOAc (3 x 100 ml). The combined organic phases are washed with 1 M HCl (4x100 ml) to remove HOSu and concentrated under reduced pressure. The obtained residue is further purified by recrystallization in EtOAc.

217

Part 3: Experimental Procedures

XIII.3.6.2 3-(9-FLUORENYLMETHYLOXYCARBONYL)AMINO-2-(4- BROMOBENZYL)PROPIONIC ACID VI.60

Yield 82% (white powder) Br

O O Formula: C25H22BrNO4

HN -1 COOH Molecular Weight: 480.35 g mol

LC-MS: Peak at 5.4 min; ES-MS positive mode [m/z (fragment, intensity)]: 482.0 (M(81Br)+H+, 25), 480.0 M(79Br)+H+, 25), 304.0 (M(81Br)- fluorenylmethyl+2H+, 20), 302.0 (M(79Br)-fluorenylmethyl+2H+, 20), 258.0 (M(81Br)- + 79 + dibenzofulvene-CO2+H , 20), 256.0 (M( Br)-dibenzofulvene-CO2+H , 20), 179.1 (fluorenyl methyl cation, 100).

1 H NMR: (500 MHz, DMSO-d6): δ 12.33 (br.s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.70 (d, J = 7.22 Hz, 2H), 7.51 (t, J = 5.3 Hz, 1H), 7.47 (d, J = 8.2 Hz, 2H), 7.42 (t, J = 7.4 Hz, 1H), 7.36-7.25 (m, 2H), 7.23-7.02 (m, 2H), 4.39-4.19 (m, 2H), 3.28-3.09 (m, 2H), 2.83-2.57 (m, 2H) ppm.

13 C NMR: (500 MHz, DMSO-d6): δ 174.9 (C), 156.6 (C), 144.3 (C), 144.2 (C), 141.2 (C), 139.1 (C), 131.6 (CH), 129.2 (CH), 128.7 (CH), 128.6 (CH), 128.1 (CH), 128.05 (CH), 127.5 (CH), 127.2 (CH), 126.6 (CH), 125.6 (CH), 125.8 (CH), 120.6 (CH), 119.8 (C), 65.9 (CH2), 47.4 (CH),

47.2 (CH), 42.4 (CH2), 34.8 (CH2) ppm.

TLC: Rf =0.42 (dichloromethane/MeOH 90/10).

XIII.3.6.3 3-(9-FLUORENYLMETHYLOXYCARBONYL)AMINO-2-(4- CHLOROBENZYL)PROPIONIC ACID VI.62

Cl Yield 92% (white powder)

O O Molecular Formula: C25H22ClNO4 HN COOH Molecular Weight: 435.89 g mol-1

LC-MS: Peak at 5.2 min; ES-MS positive mode [m/z (fragment, intensity)]: 460.0 (M(37Cl)+Na+, 11), 458.0 (M(35Cl)+Na+, 33), 438.1 (M(37Cl)+H+, 13), 436.1

218

Part 3: Experimental Procedures

(M(35Cl)+H+, 38), 260.0 (M(37Cl)-dibenzofulvene+H+, 9), 258.0 (M(37Cl)- dibenzofulvene+H+ 37 + 35 + ,27), 242.0 (M( Cl)-dibenzofulvene-H2O+H , 12), 240.0 ((M( Cl)-dibenzofulvene-H2O+H , 36), 37 + 35 + 216.0 (M( Cl)-dibenzofulvene-CO2+H , 7), 214.1 ((M( Cl)-dibenzofulvene-CO2+H , 20), 196.0 (9), 180.1 (18), 179.1 (fluorenylmethyl cation, 100).

35 + + HRMS (ESI): C25H23 ClNO4 [M+H ]: calculated 436.1316, found: 436.1305.

1 H NMR: (300 MHz, DMSO-d6): δ 12.30 (br s, 1H), 7.87 (d, J = 7.5 Hz, 2H), 7.68 (d, J = 7.3 Hz, 2H), 7.49 (t, J = 5.7, 1H), 7.40 (t, J = 7.3 Hz, 2H), 7.36-7.28 (m, 4H), 7.20 (d, J = 8.3 Hz, 1H), 7.18 (br.s, 1H), 4.37-4.17 (m, 3H), 3.30-2.60 (m, 5H) ppm.

13 C NMR: (75 MHz, DMSO-d6): δ 174.4 (C), 156.1 (C), 143.8 (C), 140.7 (C), 138.1 (C), 130.8

(C), 130.6 (CH), 128.1 (CH), 127.6 (CH), 127.0 (CH), 125.2 (CH), 120.1 (CH), 65.35 (CH2), 46.9

(CH), 46.7 (CH), 41.9 (CH2), 34.3 (CH2) ppm.

IR(HATR): 3323 (m), 3070 (vw), 3013 (vw), 2982 (w), 2956 (vw), 2930 (w), 1692 (s), 1637 (m), 1584 (vw), 1556 (m), 1492 (m), 1463 (vw), 1450 (w), 1408 (m), 1370 (m), 1326 (w), 1268 (s), 1217 (vw), 1204 (vw), 1180 (w), 1160 (m), 1110 (w), 1089 (m), 1014 (w), 983 (w), 870 (w), 845 (m), 795 (m), 756 (w), 738 (w), 729 (vw), 646 (m) cm-1.

TLC: Rf =0.51 (dichloromethane/MeOH 90/10).

XIII.3.6.4 3-(9-FLUORENYLMETHYLOXYCARBONYL)AMINO-2-(NAPHTH-2-YLMETHYL) PROPIONIC ACID VI.63

Yield: 87% (white powder)

O O M.p 192 °C HN COOH Molecular Formula: C29H25NO4

Molecular Weight: 451.51 g mol-1

LC-MS: Peak at 5.4 min; ES-MS positive mode [m/z (fragment, intensity)]: 452.2 (M+H+, 90), 274.1, ([M-dibenzofulvene+H+, 10).

+ + HRMS (ESI): C29H26NO4 [M+H ]: calculated 452.1861, found 452.1844.

219

Part 3: Experimental Procedures

1 H NMR: (500 MHz, Acetone-d6): δ 7.88-7.78 (m, 5H), 7.76 (s, 1H), 7.70-7.68 (d, J=7.4, 2H), 7.50-7.35 (m, 5H), 7.31 (t, J= 7.2 Hz, 2H), 6.6 (br.s., NH), 4.33 (d, J=7.2 Hz, 2H), 4.22 (t, J= 7.2 Hz, 1H), 3.53-3.40 (m, 2H), 3.19-2.93 (m, 3H) ppm.

13 C NMR: (125 MHz, Acetone-d6): δ 178.9 (C), 174.2 (C), 156.3 (C), 144.3 (C), 144.2 (C), 141.2 (C), 136.9 (C), 133.7 (C), 132.4 (C), 127.9 (CH), 127.6 (CH), 127.5 (CH), 127.5 (CH), 127.3 (CH),

127.0 (CH), 125.9 (CH), 125.4 (CH), 125.2 (CH), 119.9 (CH), 66.1 (CH2), 47.3 (CH), 47.2 (CH),

42.2 (CH2), 35.6(CH2) ppm.

IR(HATR): 3338 (m), 3042.9 (w), 2966 (w), 2930 (w), 1686 (s), 1615 (m), 1559 (s), 1449 (m), 1379 (w), 1361 (w), 1323 (w), 1265 (m), 1213 (w), 1290 (w), 1162 (m), 1129 (w), 1102 (w), 1083 (vw), 1021 (w), 986 (w), 960 (w), 948 (w), 895 (w), 858 (w), 812 (w), 758 (m), 740 (s),701 (vw), 645 (m) cm-1.

TLC: Rf = 0.22 (hexane/EtOAc/AcOH 70/30/1).

XIII.3.6.5 2-(9-FLUORENYLMETHYLOXYCARBONYL)AMINOMETHYL-4-METHYL PENTANOIC ACID VI.64

Yield: 84% (White fluffy powder) O O M.p. 178 °C HN COOH Molecular Formula: C22H25NO4

Molecular weight: 367.43 g mol-1

LC-MS: Peak at 5.0 min; ES-MS positive mode [m/z (fragment, intensity)]: 368.2 (M+H+, 100), 190.1 ([M-t-butyl]+2H+, 5).

+ + HRMS (ESI): C22H26NO4 [M+H ]: calculated: 368.1861, found: 368.1862.

1 H NMR (300 MHz, DMSO-d6): δ 7.88 (d, J=7.5 Hz, 2H), 7.69 (d, J=7.4, 2H), 7.50-7.25 (m, 4H), 4.35-4.15 (m, 3H), 3.25-3.00 (m, 2H), 2.60-2.46 (m, 1H), 1.63-1.14 (m, 3H), 0.87 (d, J=1.7 Hz, 3H), 0.84 (d, J=1.7 Hz, 3H) ppm.

220

Part 3: Experimental Procedures

13 C NMR (75 MHz, DMSO-d6): δ 176.0 (C), 156.2 (C), 143.9 (C), 143.9 (C), 140.7 (C), 128.9 (CH), 127.6 (CH), 127.3 (CH), 127.1 (CH), 125.2 (CH), 125.0 (CH), 121.4 (CH), 120.1 (CH), 50.4

(CH2), 46.7 (CH), 43.7 (CH), 42.8 (CH2), 37.4 (CH2) 25.7 (CH), 23.0 (CH3), 21.9 (CH3) ppm.

IR (HATR): 3287 (m), 2959 (m), 2942 (m), 2919 (m), 2908 (m), 2866 (m), 1689 (s, sharp), 1556 (m), 1465 (w), 1447 (m), 1384 (w), 1365 (w), 1330 (w), 1297 (s), 1265 (m), 1254 (m), 1234 (m), 1220 (m), 1219 (vw), 1161 (m), 1128 (w), 1108 (w), 1084 (w), 1043 (vw), 1009.5 (w), 995 (w), 938 (m), 884 (w), 813 (w), 782 (w), 756 (m), 739 (s, sharp), 672 (m), 639 (m) cm -1.

TLC: Rf = 0.30 (hexane/EtOAc/AcOH 70/30/1).

221

Part 3: Experimental Procedures

XIII.4 SYNTHESIS OF 3-SUBSTITUTED-1,5-BENZODIAZOCINE-2,6-DIONES

XIII.4.1 β2-Amino acid coupling on Wang resin

1. Fmoc-AA-OH, 2 eq DIC, 0.2 eq DMAP, CH2Cl2, r.t, 24 h O 2. Ac2O/DIPEA/CH2Cl2 (1/1/3), r.t, 2x1 hr OH O NH2 3. 20% 4-methylpiperidine in DMF, 2x20 min R1 VI.6 VI.82

General procedure: Preactivation: To a cooled (0°C) solution of N-Fmoc-β2-amino acid VI.62-

VI.64 (1 mmol, 2 eq) in CH2Cl2 (5ml), (1 mmol, 2 eq) DIC is added. The reaction mixture is stirred for 20 minutes at 0 °C.

Coupling: The crude preactivation mixture is transferred to a solid-phase reaction vessel containing Wang resin (0.5 mmol, 1 eq, calculated from manufacturer’s loading 1.9 mmol/g), washed and preswollen with CH2Cl2) after which DMAP is added (0.1 mmol, 0.2 eq). The suspension is shaken for 24 hours at room temperature after which the resin is filtered and washed consecutively with CH2Cl2 (3x), DMF (3x), MeOH (3x), and CH2Cl2 (3x).

Capping: The resin is suspended in Ac2O/DIPEA/CH2Cl2 (1/1/3, 10 ml) and shaken for 1 h. The resin is filtered and washed with CH2Cl2. This capping procedure is repeated once). The resin is filtered and consecutively washed with CH2Cl2 (3x), DMF (3x), MeOH (3x) and CH2Cl2 (3x) and dried under reduced pressure.

Loading: The loading of the resulting resins was determined by Fmoc UV quantification:

(R1=isobutyl: 0.6626 mmol/g, R1=naphtha-2-ylmethyl: 0.4785 mmol/g, R1=chlorobenzyl: 0.3137 mmol/g). Yields of final products were calculated in reference to these loading values. An amount of resin-bound Fmoc-amino acid is suspended in a 20% 4-methylpiperidine solution in DMF. The absorbance of the dibenzofulvene/4-methylpiperidine adduct at 300 nm is correlated to its concentration using a calibration line.

Deprotection: The resin is washed with DMF (3x) and treated with a solution of 20% 4- methylpiperidine in DMF for 20 min. The resin is filtered and washed with DMF (3x). The 4- methylpiperidine treatment is repeated once, then the resin is subsequently filtered and washed with DMF (3x), MeOH (3x), CH2Cl2 (3x) and DMF (3x).

222

Part 3: Experimental Procedures

-Amount of resin. For synthesis of the individual library members we used 0.4788 mmol of the above-prepared Fmoc-β-amino acid-loaded Wang resins.

XIII.4.2 Nosyl coupling

O 5 eq o-Ns-Cl, 10 eq collidine, O O O NO2 CH2Cl2, r.t, 2x1h S O NH O N 2 H R1 R1 VI.82 VI.83

General procedure: To a suspension of resin VI.82 (0.800 g, 0.478 mmol, 1 eq) in dry DCE (11.3 ml) is added 2,4,6-collidine (633 μl, 4.788 mmol, 10 eq) and 2-nitrobenzenesulfonyl chloride (531 mg, 2.394 mmol, 5 eq). After 1 hour shaking, the resin is filtered and washed with DMF (3x15 ml), MeOH (3x15 ml) and CH2Cl2 (3x15 ml). This procedure is repeated once, delivering the nosyl protected resin bound amino acid VI.83.

This reaction was optimized for compounds VI.83, with R1=2-naphthyl or isobutyl. LC-MS analysis after cleavage of a small amount of resin-bound material confirmed the identity of the expected products.

Molecular Formula: C20H18N2O6S O O O NO2 S HO N Molecular Weight: 414.43 g mol-1. H LC-MS: Peak at 4.8 min, ES-MS negative mode [m/z (fragment, intensity)]: 827.0 (2M-H+, 10), 413.0 (M-H+, 100).

Molecular Formula: C13H18N2O6S O O O NO2 S HO N Molecular Weight: 330.35 g mol-1. H LC-MS: Peak at 4.4 min, ES-MS negative mode [m/z (fragment, + - intensity)]: 329.1 (M-H , 100), 201.2 (NO2-Ph-SO2NH , 5).

223

Part 3: Experimental Procedures

XIII.4.3 Mitsunobu alkylation

O O O NO2 O O O NO2 S S O N 10 eq VI.70-VI.80, 5 eq Ph3P, O N H 5 eq DIAD, DCE, r.t, 3x2h R1 R1 R2 VI.83 VI.84

General procedure: the resin was suspended in DCE (12 ml) and triphenylphosphine (628 mg, 2.394 mmol, 5 eq), alcohol VI.70-VI.80 (4.788.mmol, 10 eq) and DIAD (471 μl, 2.394 mmol, 5 eq) were sequentially added. The reaction mixture is shaken for 2 h at room temperature after which the resin is drained and washed with dry CH2Cl2 and dry DCE. This Mitsunobu procedure was repeated twice, the resin was filtered and washed with DMF (3x15 ml), MeOH (3x15 ml) and

CH2Cl2 (3x15 ml).

This reaction was optimized for compounds VI.84, with R1=2-naphthyl, R2=Me and R1=isobutyl,

R2=Me. LC-MS analysis after cleavage of a small amount of resin-bound material confirmed the identity of the expected products.

Molecular Formula: C21H20N2O6S O O O NO2 S HO N Molecular Weight: 428.45 g mol-1. Me LC-MS: Peak at 5.0 min, ES-MS negative mode [m/z (fragment, intensity)]: 855.1 (2M-H+, 100), 427.1 (M-H+, 100).

Molecular Formula: C14H20N2O6S O O O NO2 S -1 HO N Molecular Weight: 344.38 g mol .

LC-MS: Peak at 4.5 min, ES-MS negative mode [m/z (fragment, + + intensity)]: 687.1 (2M-H , 38), 343.1 (M-H , 80), 215.1 (NO2-Ph- - SO2NMe , 28).

224

Part 3: Experimental Procedures

XIII.4.4 Nosyl removal

O O O NO2 O S 2.5 eq DBU, 5 eq HSCH CH OH O N 2 2 O NH DMF, r.t, 2x30min R1 Me R1 Me VI.85 VI.84

General procedure: The resin is suspended in DMF (12 ml) and DBU (179 μl, 1.197 mmol, 2.5 eq) and 2-mercaptoethanol (168 μl, 2.394 mmol, 5 eq) were added. The resulting suspension was shaken for 30 min after which the resin was drained and washed with DMF (3x15 ml), MeOH

(3x15 ml) and CH2Cl2 (3x15 ml). This procedure is repeated once, after which the resin was drained and washed with DMF (3x15 ml).

This reaction was optimized for compounds VI.85, with R1=2-naphthyl, R2=Me and R1=isobutyl,

R2=Me. LC-MS analysis after cleavage of a small amount of resin-bound material confirmed the identity of the expected products.

O Molecular Formula: C15H17NO2

HO NH Molecular Weight: 243.30 g mol-1. Me LC-MS: Peak at 4.1 min, ES-MS positive mode [m/z (fragment, intensity)]: 244.1 (M+H+, 100).

O Molecular Formula: C8H17NO2

HO NH Molecular Weight: 159.22 g mol-1.

LC-MS: Peak at 3.1 min, ES-MS negative mode [m/z (fragment, intensity)]: 158.2 (M-H+, 100).

225

Part 3: Experimental Procedures

XIII.4.5 Coupling of Fmoc-anthranilic acids

O O O NHFmoc 10 eq VI.7-VI.11, 5 eq DIC O NH O N

R1 R2 CH2Cl2/DMF 9/1, r.t, 24 h R1 R2 R3 VI.85 VI.86

General procedure: Preactivation: To a cooled solution (0°C) of the appropriate N-Fmoc- protected anthranilic acid derivative VI.7-VI.11 (4.788 mmol, 10 eq) in CH2Cl2/DMF (9/1) (11.3 ml), was added DIC (252 μl, 2.394 mmol, 5 eq) and the reaction mixture was stirred for 30 min at 0 °C.

Coupling: The crude preactivation mixture was transferred to the appropriate resin VI.85, preswollen in CH2Cl2/DMF 9/1, and the resulting suspension is shaken for 24 h. The resin was drained and washed consecutively with DMF (3x15 ml), MeOH (3x15 ml) and CH2Cl2 (3x15 ml).

This reaction was optimized for compounds VI.86, with R1=2-naphthyl, R2=Me, R3=H and R1=2- naphthyl, R2=Me, R3=H. LC-MS analysis after cleavage of a small amount of resin-bound material confirmed the identity of the expected products.

O O NHFmoc Molecular Formula: C37H32N2O5 HO N Molecular Weight: 584.66 g mol-1. Me LC-MS: Peak at 5.6 min, ES-MS positive mode [m/z (fragment, intensity)]: 585.2 (M+H+, 100).

Molecular Formula: C H N O O O NHFmoc 30 32 2 5 HO N Molecular Weight: 500.58 g mol-1. Me LC-MS Peak at 5.6 min, ES-MS positive mode [m/z (fragment, + + intensity)]: 501.2 (M+H , 100), 160.2 (HOOC-CH(iBu)-CH2-NHMe+H , 28).

226

Part 3: Experimental Procedures

X.4.6 Fmoc removal

O O NHFmoc O O NH2 20 % 4-methylpiperidine in DMF O N O N R1 R2 r.t, 2x20min R R R 1 2 VI.86 3 VI.87 R3

Removal of the Fmoc protecting group, See section X.4.1

X.4.7 Cleavage from the resin

O O NH2 O O NH2 TFA/H2O 9/1, 1 h. O N HO N

R1 R2 R1 R2 R3 R3 VI.87 VI.88

General procedure: resin VI.87 is suspended in a mixture of trifluoroacetic acid/water 95/5 (15 ml) and shaken for 1 hour at room temperature. The resin is filtered and washed extensively with dichloromethane. The filtrate is concentrated under reduced pressure and the obtained residue is further purified by flash chromatography (F.C) or preparative reversed-phase HPLC.

XIII.4.7.1 4-METHYL-2-(METHYL-(N-ANTHRANOYL-N-METHYL)PENTANOIC ACID XIII.1

F.C.: hexane/EtOAc/AcOH 60/40/1 O O NH2 HO N SPS yield: 88% (colorless glass), 46 mg from 119 mg resin VI.82

Molecular Formula: C15H22N2O3

Molecular weight: 278.34 g mol-1.

LC-MS: Peak at 3.7 min; ES-MS positive mode [m/z (fragment, intensity)]: 579.2 (2M+Na+, 8), 279.1 (M+H+, 100), 261.1 (M-OH-, 10).

HRMS (ESI): Not detectable by mass spectrometer (positive and negative mode).

227

Part 3: Experimental Procedures

1 H NMR: (300 MHz, CD3OD): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.11 (td, J= 1.3/7.6 Hz, 1H), 7.01 (d, J=6.9 Hz ,1H), 6.74 (d, J=8.1 Hz, 1H), 6.65 (t, J= 7.4 Hz, 1H), 3.61 (br.s, 2H), 2.98 (s, 3H), 2.89-2.51 (m, 1H), 1.79-1.09 (m, 3H), 0.89 (br. s, 6H) ppm.

13 C NMR: (125 MHz, CD3OD): no multiple conformers observed: δ 146.1 (C), 131.7 (CH), 128.7

(CH), 122.1 (C), 118.3 (CH), 117.5 (CH), 50.0 (CH2), 44.1 (CH), 40.6 (CH2), 37.1 (CH3), 27.5

(CH), 23.6 (CH3), 22.5 (CH3) ppm.

IR (HATR): 3380 (m), 3345 (m), 3166 (w), 3070 (w), 2953 (m), 2926 (m), 1711 (m), 1614 (s), 1584 (s), 1538 (s), 1491 (m), 1465 (m), 1450 (m), 1383 (w), 1366 (w), 1353 (w), 1315 (m), 1301 (m), 1261 (m), 1197 (s), 1158 (m), 1123 (m), 1081 (w), 1031 (w), 998 (w), 942 (vw), 893 (w), 852 (vw), 816 (w), 792 (w), 745 (s), 699 (w), 660 (w), 622 (w) cm-1.

TLC: Rf = 0.41 (hexane/EtOAc/AcOH 40/60/1).

XIII.4.7.2 2-(N(2-AMINO-6-FLUOROBENZOYL),N(METHOXYETHOXY-2- ETHYL)AMINOMETHYL)PENTANOIC ACID XIII.2

F.C.: hexane/EtOAc/AcOH 60/40/10 O O NH2 HO N SPS yield: 53% (brown glass), 93 mg from 800 mg resin VI.82

F Molecular Formula: C19H29FN2O5 O O Molecular weight: 384.44 g mol-1.

LC-MS: Peak at 4.1 min; ES-MS positive mode [m/z (fragment, intensity)]: 385.1 (M+H+, 100).

+ + HRMS (ESI): C19H30FN2O5 [M+H ] calculated: 385.2138, found: 385.2142.

1 H NMR: (300 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.13-6.95 (m, 1H), 6.57-6.26 (m, 4H), 4.21-2.51 (m, 14H), 1.73-0.96 (m, 3H), 0.91 (d, J= 6.4 Hz, 3.74H), 0.81 (d, J= 6.2 Hz, 0.66H), 0.79 (d, J= 6.2 Hz, 0.66H), 0.72 (d, J= 6.2 Hz, 0.74H), 0.60 (d, J= 6.2 Hz, 0.47H) ppm.

228

Part 3: Experimental Procedures

13 C NMR: (75 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): major conformer δ 179.5 (d, J= 21.0 Hz, C), 178.4 (CO), 166.9 (CO), 158.5 (d, J= 244.3 Hz, C), 145.7 (d, J= 22.2 Hz, C), 131.1 (d, J= 22.0 Hz, CH), 111.6 (CH), 104.0 (CH),

71.6 (CH2), 70.1 (CH2), 68.6 (CH2), 58.8 (CH3), 49.8 (CH2), 44.5 (CH2), 42.2 (CH), 39.1 (CH2),

25.9 (CH), 22.9 (CH3), 22.0 (CH3) ppm.

IR (HATR): 3443 (vw), 3355 (w), 2953 (w), 2869 (w), 1715 (m), 1621 (s), 1614 (s), 1463 (s), 1434 (m), 1385 (w), 1367 (w), 1352 (w), 1327 (w), 1245 (m), 1196 (s), 1113 (s), 1095 (s), 1050 (m), 1029 (w), 993 (m), 923 (w), 842 (w), 789 (m), 767 (w), 753 (m), 720 (m), 641 (w) cm-1.

TLC: Rf = 0.05 (hexane/EtOAc/AcOH 60/40/1).

XIII.4.7.3 4-METHYL-2-(N-(4-PHENYLBENZYL),N(2-AMINO-5- METHOXYBENZOYL)AMINOMETHYL)PENTANOIC ACID XIII.3

F.C.: hexane/EtOAc/AcOH 60/40/1 O O NH2 HO N SPS yield: 31% (brown powder), 127 mg from 800 mg resin VI.82

OMe Molecular Formula: C28H32N2O4

Molecular weight: 460.56 g mol-1.

LC-MS: Peak at 5.3 min; ES-MS positive mode [m/z (fragment, intensity)]: 921.4 (2M+H+, 30), 461.2 (M+H+, 100).

+ + HRMS (ESI): C28H33N2O4 [M+H ] calculated: 461.2440, found: 461.2462.

1 H NMR: (300 MHz, CDCl3): (two amide rotamers ratio 80/20) δ 7.57 (d, J=7.5 Hz, 2H), 7.47- 7.42 (t, J=7.0 Hz, 2H), 7.39-7.33 (tt, J=1.3/7.4 Hz, 2H), 7.25-7.11 (m, 4H), 6.74 (d, J=11.4 Hz, 2H), 5.12 (br.s, 0.2H), 4.59 (br.s, 0.8H), 3.91-3.82 (m, 1H), 3.58 (s, 3H), 3.39-3.30 (m, 1H), 3.02- 2.70 (m, 1H), 1.64-1.08 (m, 3H), 0.87-0.58 (br.s, 6H) ppm.

13 C NMR: (75 MHz, CDCl3): no multiple conformers observed: δ 179.0 (CO), 171.4 (CO), 162.7 (C), 140.7 (C), 140.4 (C), 135.2 (C), 128.8 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.4 (CH), 127.3 (CH), 126.9 (CH), 122.7 (C), 119.4 (CH), 119.5 (CH), 118.3 (C), 117.3 (CH), 117.2 (CH),

229

Part 3: Experimental Procedures

111.9 (CH), 55.5 (CH3), 52.9 (CH2), 47.7 (CH2), 42.0 (CH), 39.0 (CH2), 25.9 (CH), 22.7 (CH3),

22.0 (CH3) ppm.

IR (HATR): 3447 (vw), 3559 (vw), 2952 (w), 1697 (m), 1633 (m), 1591 (m), 1499 (m), 1485 (m), 1465 (m), 1426 (m), 1364 (w), 1281 (m), 1200 (s), 1135 (s), 1075 (w), 1037 (m), 1007 (w), 928 (vw), 816 (w), 797 (w), 759 (m), 739 (w), 721 (m), 696 (m) cm-1.

TLC: Rf = 0.29 (hexane/EtOAc/AcOH 60/40/1).

XIII.4.7.4 4-METHYL-2-(N-ANTHRANOYL,N-(3- METHOXYBENZYL)AMINOMETHYL)PENTANOIC ACID XIII.4

F.C.: hexane/EtOAc/AcOH 80/20/1 O O NH2 HO N SPS yield: 62% (pale brown), 109 mg from 800 mg resin VI.82

Molecular Formula: C22H28N2O4

Molecular weight: 384.46 g mol-1. MeO LC-MS: Peak at 4.6 min; ES-MS positive mode [m/z (fragment, intensity)]: 769.3 (2M+H+, 10), 385.1 (M+H+).

+ + HRMS (ESI): C22H29N2O4 [M+H ] calculated: 385.2127, found: 385.2118.

1 H NMR: (300 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.26-7.08 (m, 3H), 6.85-6.60 (m, 7H), 4.62-4.44 (m, 2H), 3.78 (s, 3H), 3.70- 3.34 (m, 2H), 3.08-2.80 (m, 1H), 1.62-1.40 (m, 2H), 1.23-1.03 (m, 1H), 0.87 (br. s, 6H) ppm.

13 C NMR: (75 MHz, CDCl3): no multiple conformers observed: δ 179.3 (CO), 172.1 (CO), 160.0 (C), 144.2 (C), 138.0 (C), 130.6 (CH), 129.7 (CH), 127.6 (CH), 120.3 (C), 119.7 (CH), 117.9 (CH),

116.6 (CH), 113.2 (CH), 112.9 (CH), 55.2 (CH3), 53.8 (CH2), 47.7 (CH2), 42.1 (CH), 39.1 (CH2),

25.9 (CH), 22.7 (CH3), 22.03 (CH3) ppm.

IR (HATR): 3596 (vw), 3357 (vw), 2952 (w), 1718 (m), 1597 (s), 1585 (s), 1490 (s), 1465 (s), 1421 (s), 1382 (vw), 1367 (w), 1287 (m), 1260 (s), 1191 (s), 1191 (s), 1149 (s), 1116 (m), 1047 (m), 1036 (m), 994 (w), 971 (w), 873 (vw), 775 (m), 748 (s), 691 (w), 623 (vw) cm-1.

230

Part 3: Experimental Procedures

TLC: Rf = 0.18 (hexane/EtOAc/AcOH 60/40/1).

XIII.4.7.5 4-METHYL-2-(METHYL-(N-ANTHRANOYL,N-(1-PHENYLPROP-1-EN-3- YL)AMINOMETHYL)PENTANOIC ACID XIII.5

F.C.: hexane/EtOAc/AcOH 80/20/1 to 70/30/1 O O NH2 HO N SPS yield: 38% (brown glass), 66 mg from 800 mg resin VI.82

Molecular Formula: C23H28N2O3

Molecular weight: 380.48 g mol-1.

LC-MS: Peak at 4.9 min; ES-MS positive mode [m/z (fragment, intensity)]: 761.3 (2M+H+, 24), 381.3 (M+H+, 100).

+ + HRMS (ESI): C23H29N2O3 [M+H ] calculated: 381.2178, found: 381.2175.

1 H NMR: (300 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.30-7.01 (m, 7H), 6.68-6.53 (m, 4H), 6.35 (d, J =14.1 Hz, 1H), 5.95 (d, J=12.6 Hz, 1H), 4.10-3.86 (m, 2H), 3.69-3.40 (m, 2H), 3.03-2.82 (m, 1H), 1.69-1.29 (m, 2H), 1.23-1.07 (m, 1H), 0.70-1.01 (m, 6H) ppm.

13 C NMR: (75 MHz, CDCl3): no multiple conformers observed: δ 179.1 (CO), 171.9 (CO), 143.9 (C), 136.1 (C), 133.1 (CH), 130.5 (CH), 128.7 (CH), 128.5 (CH), 128.1 (CH), 127.8 (CH), 127.3

(CH), 126.4 (CH), 124.2 (CH), 120.6 (C),117.7 (CH), 116.5 (CH), 52.1 (CH2), 48.0 (CH2), 42.3

(CH), 39.1 (CH2), 25.9 (CH), 22.8 (CH3), 22.0 (CH3) ppm.

IR (HATR): 3447 (vw), 3348 (vw), 2951 (w), 2868 (w), 1718 (m), 1613 (s), 1586 (s), 1465 (s), 1444 (m), 1366 (m), 1238 (m), 1193 (s), 1157 (m), 1116 (w), 1051 (w), 1029 (w), 962 (m), 801 (vw), 748 (s), 691 (m), 638 (w) cm-1.

TLC: Rf = 0.21 (hexane/EtOAc/AcOH 60/40/1).

231

Part 3: Experimental Procedures

XIII.4.7.6 3-(N-ANTHRANOYL-N-METHYL)AMINO-2-(NAPHTH-2-YLMETHYL)PROPIONIC ACID XIII.6

F.C.: hexane/EtOAc/AcOH 40/60/1 O O NH2 HO N HPLC: method A, tret = 11.7 SPS yield 37% (colorless glass) 60 mg from 800 mg resin VI.82

Molecular Formula: C22H22N2O3

Molecular weight: 362.42 g mol-1.

LC-MS: Peak at 4.2 min; ES-MS positive mode [m/z (fragment, intensity)]: 725.2 (2M+H+, 10), 363.1 (M+H+, 100).

+ + HRMS (ESI): C22H23N2O3 [M+H ] calculated: 363.1708, found: 363.1695.

1 H NMR: (300 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.84-7.68 (m, 3H), 7.67-7.59 (m, 1H), 7.49-7.39 (m, 2H), 7.30-7.17 (m, 1H), 7.15-6.92 (m, 2H), 6.71-6.51 (m, 2H), 5.3 (br.s, 2H), 4.01 (br.s, 1H), 3.46 (dd, J= 3.6/13.5 Hz, 1H), 3.28-3.15 (m, 2H), 2.80-3.03 (m, 4H) ppm.

13 C NMR: (75 MHz, CDCl3): no multiple conformers observed: δ 177.1 (CO), 171.8 (CO), 144.2 (C), 135.6 (C), 133.4 (C), 132.2 (C), 131.1 (CH), 130.6 (CH), 128.2 (CH), 127.5 (CH), 127.3 (CH),

127.0 (CH), 126.0 (CH), 125.5 (CH), 120.3 (C), 117.7 (CH), 116.8 (CH), 116.7 (CH), 49.1 (CH2),

45.5 (CH), 36.1 (CH2), 35.8 (CH3) ppm.

IR (HATR): 3462 (w), 3369 (w), 3229 (w), 3033 (m), 2937 (m), 1723 (m), 1710 (m), 1692 (m), 1679 (w), 1611 (s), 1586 (s), 1529 (vw), 1493 (w), 1484 (w), 1462 (w), 1449 (w), 1431 (w), 1402 (m), 1367 (w), 1346 (w), 1310 (w), 1289 (w), 1269 (w), 1201 (w), 1170 (w), 1075 (m), 1031 (vw), 948 (vw), 855 (w), 818 (m), 749 (s), 708 (vw), 630 (vw) cm-1.

TLC: Rf = 0.24 (hexane/EtOAc/AcOH 40/60/1).

232

Part 3: Experimental Procedures

XIII.4.7.7 3-(N-(2-AMINO-5-BROMOBENZOYL),N-ETHYL)AMINO-2-(NAPHTH-2-YL) PROPIONIC ACID XIII.7

F.C.: hexane/EtOAc/AcOH 80/20/1 to 70/30/1 O O NH 2 SPS yield: 63% (beige glass), 138 mg from 800 mg resin VI.82 HO N

Molecular Formula: C23H23BrN2O3 Br Molecular weight: 455.34 g mol-1.

LC-MS: Peak at 4.7 min; ES-MS negative mode [m/z (fragment, intensity)]: 909.0 (2M(Br79/Br81)- H+, 100). 456.0 (M(Br81)-H+, 20)

79 - + HRMS (ESI): C23H22 BrN2O3 [M-H ] calculated: 453.0813, found: 453.0825.

1 H NMR: (300 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.86-7.72 (m, 3H), 7.64 (s, 1H), 7.51-7.40 (m, 2H), 7.38-7.24 (m, 1H), 7.18- 7.08 (m, 2H), 6.38 (d, J=8.4 Hz, 1H), 6.22 (br.s, 2H), 3.86 (br.s, 1H), 3.48 (br.s, 1H), 3.29-3.06 (m, 4H), 2.89 (br.s, 1H), 0.93 (br.s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): no multiple conformers observed: δ 177.5 (CO), 170.2 (CO), 142.7 (C), 135.4 (C), 133.4 (C), 133.0 (CH), 132.2 (C), 129.4 (CH), 128.2 (CH), 127.9 (CH), 127.6 (CH),

127.5 (CH), 127.3 (CH), 126.9 (CH), 126.1 (CH), 122.4 (C), 118.1 (CH), 109.2 (C), 48.1 (CH2),

46.9 (CH2), 45.5 (CH), 36.1 (CH2), 13.6 (CH3) ppm.

IR (HATR): 3360 (w), 3054 (vw), 2970 (w), 2928 (w), 1713 (m), 1600 (s), 1581 (s), 1504 (w), 1483 (s), 1454 (m), 1434 (m), 1401 (m), 1382 (w), 1360 (w), 1279 (s), 1254 (s), 1170 (m), 1150 (m), 1100 (m), 1079 (m), 1014 (w), 980 (w), 953 (w), 889 (w), 854 (w), 801 (s), 746 (s), 638 (vw), 618 (w) cm-1.

TLC: Rf = 0.15 (hexane/EtOAc/AcOH 60/40/1).

233

Part 3: Experimental Procedures

XIII.4.7.8 3-(N-(4-BUT-1-YNYL),N-(2-AMINO-5-METHOXBENZOYL))AMINO-2-(NAPHTH-2- YL)PROPIONIC ACID XIII.8

F.C.: hexane/EtOAc/AcOH 70/30/1 O O NH2 HO N SPS yield: 35% (brown glass), 74 mg from 800 mg resin VI.82

Molecular Formula: C26H26N2O4 OMe Molecular weight: 430.49 g mol-1.

LC-MS: Peak at 4.5 min; ES-MS positive mode [m/z (fragment, intensity)]: 861.3 (2M+H+, 25), 431.2 (M+H+, 100).

+ + HRMS (ESI): C26H27N2O4 [M+H ] calculated: 431.1970, found: 431.1966.

1 H NMR: (300 MHz, CD3OD): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.78-7.56 (m, 4H), 7.45-7.30 (m, 3H), 6.93-6.46 (m, 3H), 3.79-3.40 (m, 7H), 3.23-2.66 (m, 3H), 2.47-2.22 (m, 3H) ppm.

13 C NMR: (75 MHz, CD3OD): no multiple conformers observed: δ 177.8 (CO), 173.5 (CO), 153.4 (C), 138.7 (C), 137.4 (C), 135.0 (C), 133.8 (C), 129.2 (CH), 128.7 (CH), 128.5 (CH), 128.4 (CH), 128.2 (CH), 127.1 (CH), 126.6 (CH), 123.1 (C), 119.2 (CH), 118.4 (CH), 113.4 (CH), 71.9 (CH),

56.3 (CH3), 56.1 (CH2), 47.0 (CH2), 37.6 (CH), 36.7 (CH2), 17.3 (CH2) ppm.

TLC: Rf = 0.08 (hexane/EtOAc/AcOH 60/40/1).

XIII.4.7.9 2-(NAPHTH-2-YL)-3-(N-(4-FLUOROBENZYL),N-(2-AMINO-5- METHYLBENZOYL))AMINOPROPIONIC ACID XIII.9

F.C.: hexane/EtOAc/AcOH 80/20/1 to 60/40/1 O O NH2 HO N SPS yield: 74% (pale brown glass), 88 mg from 800 mg resin VI.82

Molecular Formula: C29H27FN2O3 F Molecular weight: 470.53 g mol-1.

234

Part 3: Experimental Procedures

LC-MS: Peak at 5.2 min; ES-MS positive mode [m/z (fragment, intensity)]: 941.3 (2M+H+, 10), 471.3 (M+H+, 100).

+ + HRMS (ESI): C29H28FN2O3 [M+H ] calculated: 471.2084, found: 471.2076.

1 H NMR: (300 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.91-7.75 (m, 3H), 7.63 (s, 1H), 7.54-7.40 (s, 2H), 7.35-6.97 (m, 5H), 6.90 (dd, J= 8.2/1.4 Hz, 1H), 6.79 (s, 1H), 6.64 (d, J = 8.1 Hz, 1H), 4.49 (br.s, 2H), 3.61 (br.s, 1H), 3.41- 3.31 (m, 2H), 3.16 (br.s, 1H), 2.88-2.73 (m, 1H), 2.09 (s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): no multiple conformers observed: δ 175.1 (CO), 170.7 (CO), 162. (d, J =243 Hz, C), 142.6 (C), 136.4 (C), 132.9 (C), 131.7 (C), 130.6 (CH), 129.5 (CH), 129.4 (CH), 129.4 (CH), 129.4 (CH), 127.7 (CH), 127.4 (d, J=9 Hz, CH), 127.3 (CH), 126.8 (CH), 126.0 (CH),

125.4 (CH), 124.3 (C), 120.0 (C), 115.7 (CH), 115.3 (d, J=21.4 Hz, CH), 51.2 (CH2), 46.1 (CH2),

44.9 (CH), 35.6 (CH2), 19.8 (CH3) ppm.

IR (HATR): 3434 (w), 3358 (w), 2926 (w), 1737 (s), 1713 (m), 1584 (s), 1540 (s), 1505 (s), 1480 (m), 1440 (m), 1415 (m), 1347 (w), 1287 (m), 1224 (s), 1206 (s), 1170 (m), 1154 (m), 1097 (m), 1026 (m), 971 (w), 945 (w), 929 (w), 889 (vw), 852 (m), 827 (s), 810 (s), 767 (w), 748 (m), 715 (vw), 632 (w), 658 (vw) cm-1.

TLC: Rf = 0.17 (hexane/EtOAc/AcOH 60/40/1).

XIII.4.7.10 2-(4-CHLOROPHENYL)-3-(N-(2-AMINO-5-METHOXYBENNOYL),N- METHYL)AMINOPROPIONIC ACID XIII.10

F.C.: hexane/EtOAc/AcOH 40/60/1

O O NH2 HPLC: method B, tret = 17.2 HO N SPS yield: 35% (white glass), 42 mg from 1 gm resin VI.82 OMe Cl Molecular Formula: C19H21ClN2O4

Molecular weight: 376.83 g mol-1.

235

Part 3: Experimental Procedures

LC-MS: Peak at 4.1 min; ES-MS positive mode [m/z (fragment, intensity)]: 419.0 35 + 37 + 35 + (M( Cl)+CH3CN+H , 7), 379.0 (M( Cl)+H , 40), 377.0 (M( Cl)+H , 100).

35 + + HRMS (ESI): C19H22 ClN2O4 [M+H ] calculated: 377.1268, found: 377.1263.

1 H NMR: (500 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio ~ 60/40): δ 7.22-6.99 (m, 4H), 6.79 (d, J=6.3 Hz, 1.27H), 6.75-6.66 (m, 0.73H), 6.71-6.52 (m, 2H), 5.12 (br.s, 2H), 3.96-3.77 (m, 1H), 3.72 (s, 3H), 3.47-3.38 (m, 2H), 3.17-2.99 (m, 2H), 2.94 (s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): no multiple conformers observed: δ 175.7 (CO), 172.0 (CO), 141.2 (C), 132.7 (C), 132.4 (C), 130.17 (CH), 130.15 (CH), 130.09 (CH), 129.7 (CH), 128.6 (CH), 128.5

(C), 127.1 (C), 117.5 (CH), 112.4 (CH), 57.1 (CH3), 55.7 (CH), 50.5 (CH), 38.0 (CH3), 35.2 (CH2) ppm.

TLC: Rf = 0.08 (hexane/EtOAc/AcOH 60/40/1).

XIII.4.7.11 2-(4-CHLOROPHENYL)-3-(N-(2-AMINO-5-BROMOBENZOYL),N- BENZYL)AMINOPROPIONIC ACID XIII.11

F.C.: hexane/EtOAc/AcOH 80/20/1 O O NH2

HO N HPLC: method C, tret = 18.5

SPS yield: 50% (white glass), 65 mg from 1 gm resin VI.82 Br Cl Molecular Formula: C24H22BrClN2O3

Molecular weight: 501.81 g mol-1.

LC-MS: Peak at 5.1; ES-MS min positive mode [m/z (fragment, intensity)]: 505.0 (M(79Br)(37Cl)+H+, 28), 502.9 (M(79Br)(35Cl)+H+, 100).

79 35 + + HRMS (ESI): C24H23 Br ClN2O3 [M+H ] calculated: 501.0580, found: 501.0581.

1 H NMR: (500 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio ~ 70/30): δ 7.97-7.49 (m, 11H), 6.57 (d, J= 8.5 Hz, 0.72H), 6.65 (d, J= 8.3 Hz, 0.28H), 4.67-4.46 (m, 2H), 3.71-3.43 (m, 2H), 3.34-3.11 (m, 1H), 2.98-2.68 (m, 2H) ppm.

236

Part 3: Experimental Procedures

13 C NMR: (125 MHz, CDCl3): no multiple conformers observed: δ 177.2 (CO), 170.7 (CO), 143.3 (C), 133.5 (CH), 132.5 (C), 130.1 (CH), 128.9 (CH), 128.7 (CH), 128.0 (CH), 127.6 (CH), 121.8

(C), 118.2 (CH), 109.3 (C), 48.0 (CH2), 47.5 (CH2), 45.4 (CH), 35.2 (CH2) ppm.

TLC: Rf = 0.19 (hexane/EtOAc/AcOH 70/30/1).

237

Part 3: Experimental Procedures

XIII.4.8 Cyclization in solution

O O O NH H 2 N DCC-PS, CH Cl , 1h, R1 HO N 2 2 r.t R3 R R 1 2 N R3 O R2 VI.88 VI.1

To a solution of purified α,ω-β-amino carboxylic acids (0.197 mmol, 1 eq) in CH2Cl2 (19.7 ml) is added polystyrene-bound DCC (loading 2.3 mmol/g, 0.568 mmol, 2.9 eq). The obtained suspension is shaken for 1 h at room temperature after which the resin was filtered and washed with CH2Cl2. The filtrate was concentrated under reduced pressure. The obtained crude compounds are purified by flash chromatography (F.C) or preparative reversed-phase HPLC.

XIII.4.8.1 3,4-DIHYDRO-3-ISOBUTYL-5-METHYLBENZO[g][1,5]DIAZCOCINE-2(1H),6(5H)- DIONE VI.89

F.C.: Hexane/EtOAc/AcOH 40/60/10 O HN CYC yield: 67% (colorless glass), 18 from 23 mg XIII.1

N Molecular Formula: C15H20N2O2 O Molecular weight: 260.33 g mol-1.

LC-MS: Peak at 4.9 min; ES-MS positive mode [m/z (fragment, intensity)]: 521.2 (2M+H+, 100), 261.1 (M+H+, 38).

1 HNMR: (300 MHz, Acetone-d6): presence of two inversed boat-type conformers (ratio 80/20 individual assignments not possible): δ 8.70-8.30 (br.m, NH), 7.55-7.30 (m, 3H), 7.24 (d, J =7.7 Hz, 1H), 3.75-3.50 (m, 0.25H), 3.45-3.13 (m, 2.45H), 3.09 (s, ~2.35H), 3.02 (br.s, ~0.65H), 2.70- 2.59 (m, 0.25H), 1.70-1.56 (m, 1H), 1.37-1.25 (m, 1H), 1.13-1.03 (m, 1H), 0.88 (d, J= 1.3 Hz, ~2.4H), 0.87 (d, J= 1.7 Hz, ~2.5H), 0.80 (br.d, J= 6.2 Hz, ~0.7H), 0.73 (br.d, J= 5.6 Hz, ~0.5H) ppm.

238

Part 3: Experimental Procedures

13 CNMR: (75 MHz, Acetone-d6): no multiple conformations observed: δ 173.8 (CO), 131.8 (CH),

128.9 (CH), 128.8 (CH), 128.0 (CH), 54.2 (CH2), 47.4 (CH3), 33.5 (CH), 29.8 (CH2), 26.9 (CH),

23.6 (CH3), 23.0 (CH3) ppm.

TLC: Rf = 0.15 (Hexane/EtOAc/AcOH 40/60/10).

XIII.4.8.2 3,4-DIHYDRO-7-FLUORO-3-ISOBUTYL-5-(2-METHOXYETHOXY-2- ETHYL)BENZO [g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VI.90

O F.C.: hexane/EtOAc/AcOH 70/30/10 HN CYC yield: 55% (white glass), 40 mg from 76 mg XIII.2

N HPLC: method C, tret = 17.9 min and 18.9 min, mixture of F O O O diastereomeric atropisomers

Molecular Formula: C19H27FN2O4

Molecular weight: 366.42 g mol-1.

LC-MS: Two peaks at 5.2 min and 5.3 min diastereomeric atropisomers, ratio 82/18; ES-MS positive mode [m/z (fragment, intensity)]: 367.1 (M+H+, 100).

+ + HRMS (ESI): Peak for C19H28FN2O4 [M+H ]: calculated 367.2033, found 367.2028.

1 H NMR: (500 MHz, Acetone-d6): presence of two inversed boat-type conformers (ratio 82/18, individual assignment not possible): δ 8.78-8.62 (2x br.s, NH, ratio 2/8), 7.55-7.49 (m, 1H), 7.23- 7.16 (m, 1H), 7.14-7.09 (m,1H), 3.83-3.46 (m, 9H), 3.28-3.18 (m, 5H), 1.67-150 (m, 1H), 1.31- 1.21 (m, 1H), 1.15-1.01 (m, 1H), 0.88 (d, J= 6.7 Hz, ~2.5H), 0.86 (d, J= 6.9 Hz, ~2.4H), 0.79 (d, J= 6.7 Hz, ~0.6H), 0.72 (d, J= 6.0 Hz, ~0.6H) ppm.

13 C NMR: (125 MHz, Acetone-d6): two conformers are present: major conformer: δ 173.5 (CO), 164.3 (CO), 159.6 (d, J=248.9 Hz, C), 136.2 (C), 132.0 (d, J= 9.1 Hz, CH), 123.2 (d, J= 3.6 Hz,

CH), 115.3 (d, J= 21.8 Hz, CH), 72.5 (CH2), 70.9 (CH2), 70.1 (CH2), 53.1 (CH2), 47.7 (CH3), 46.2

(CH2), 40.6 (CH2), 26.3 (CH), 23.0 (CH3), 22.4 (CH3) ppm.

239

Part 3: Experimental Procedures

IR (HATR): 3462 (w), 2953 (m), 2924 (m), 2870 (m), 1649 (s), 1613 (s), 1578 (w), 1470 (m), 1450 (w), 1420 (m), 1420 (m), 1394 (w), 1367 (w), 1338 (w), 1308 (w), 1274 (w), 1232 (w), 1198 (w), 1098 (s), 1058 (m), 1045 (m), 993 (w), 879 (vw), 824 (w), 760 (m), 715 (w), 677 (w), 613 (vw), 575 (vw) cm-1.

TLC: Rf = 0.09 (Hexane/EtOAc/AcOH 1/1/1).

XIII.4.8.3 3,4-DIHYDRO-3-ISOBUTYL-8-METHOXY-5-(4- PHENYLBENZYL)BENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VI.91

O F.C.: dichloromethane/MeOH 99/1. HN CYC yield: 30% (yellow glass), 22 mg from 80 mg XIII.3

MeO N Molecular Formula: C28H30N2O3 O Molecular weight: 442.54 g mol-1.

LC-MS: peak at 6.9 min positive mode; ES-MS [m/z (fragment, intensity)]: 885.3 (2M+H+, 100), 443.2 (M+H+, 40).

+ + HRMS (ESI): Peak for C28H31N2O3 [M+H ]: calculated 443.2334, found 443.2338.

1 H NMR: (500 MHz, Acetone-d6): presence of two inversed boat-type conformers (ratio 80/20, individual assignment not possible): δ 8.50 and 8.35 (2x br.s, ratio 2/8, NH), 7.67-7.64 (m, 4H), 7.51-7.45 (m, 4H), 7.37-7.34 (m, 1H), 7.21 (d, J=8.7 Hz, 1H), 7.08-7.01 (m, 2H), 5.32 (d, J=14.8, ~0.8H), 5.30 (d, J=14.8, ~0.2H), 4.29 (d, J=14.8 Hz, ~0.8 H), 4.09 (d, J=15.0 Hz, ~0.2 H), 3.86 (s, 3H), 3.56 (dd, J=9.7/14.7 Hz, ~0.2H), 3.32 (dd, J=13.8/6.9 Hz, ~0.8H), 3.16-2.90 (m, 2H), 1.63-1.49 (m, 1H), 1.31 (ddd, J=14.0/8.6/6.0 Hz, 1H), 1.05 (ddd, J=14.0/8.1/6.3 Hz, 1H) 0.85 (d, J= 6.3 Hz, ~2.4H), 0.84 (d, J= 6.4 Hz, ~2.5H), 0.78 (br.d, J= 6.6 Hz, ~0.6H), 0.72 (br.d, J= 6.7 Hz, ~0.6H) ppm.

13 C NMR: (125 MHz, Acetone-d6): two conformers are present: major conformer: δ 173.7 (CO), 169.6 (CO), 159.8 (C), 141.6 (C), 141.1 (C), 137.9 (C), 137.6 (C), 129.8 (CH), 129.8 (CH), 129.3

(CH), 128.3 (CH), 128.0 (CH), 127.8 (CH), 127.3 (C), 117.1 (CH), 113.2 (CH), 56.1 (CH3), 50.8

(CH2), 48.5 (CH2), 46.6 (CH), 40.6 (CH2), 26.4 (CH), 23.1 (CH3), 22.6 (CH3) ppm.

240

Part 3: Experimental Procedures

IR (HATR): 3452 (w), 3028 (m), 2953 (m), 2869 (w), 1643 (s), 1487 (s), 1470 (s), 1434 (m), 1407 (m), 1366 (w), 1319 (w), 1292 (m), 1219 (s), 1176 (w), 1118 (vw), 1097 (w), 1033 (w), 1007 (w) , 928 (vw), 857 (vw), 822 (vw), 760 (m), 739 (w), 696 (m), 638 (vw), 623 (m) cm-1.

TLC: Rf = 0.28 (Dichloromethane/MeOH 97/3).

XIII.4.8.4 3,4-DIHYDRO-3-ISOBUTYL-5-(3-METHOXYBENZYL)BENZO[g][1,5]DIAZOCINE- 2(1H),6(5H)-DIONE VI.92

O F.C.: Dichloromethane/MeOH 99/1 HN HPLC: method A, tret = 13.2

N CYC yield: 41% (brown glass), 30 mg from 79 mg XIII.4 O

Molecular Formula: C22H26N2O3

OMe Molecular weight: 366.45 g mol-1.

LC-MS: peak at 6.1 min; ES-MS positive mode [m/z (fragment, intensity)]: 733.3 (2M+H+, 100), 367.1 (M+H+, 40).

+ + HRMS (ESI): Peak for C22H27N2O3 [M+H ]: calculated 367.2021, found 367.2011.

1 H NMR: (500 MHz, Acetone-d6): presence of two inversed boat-type conformers (ratio 80/20, individual assignment not possible): δ 8.72 and 8.53 (2x br.s, NH, ratio 2/8), 7.53-7.40 (m, 3H), 7.26 (t, J= 7.1 Hz, 2H), 6.97-6.94 (m, 2H), 6.86-6.84 (m, 1H), 5.20 (d, J=14.8 Hz, ~0.8H), 5.14 (d, J=15.3 Hz, ~0.2H), 4.32 (d, J=14.8 Hz, ~0.8H), 4.15 (d, J=14.5 Hz, ~0.2H), 3.80 (s, 3H), 3.60- 3.24 (m, 1H), 3.11-2.60 (m, 2H), 1.90-1.00 (m, 3H), 0.83 (d, J= 6.0 Hz, ~2.3H), 0.81 (d, J= 6.1 Hz, ~2.6H), 0.76 (d, J= 6.3 Hz, ~0.6H), 0.69 (d, J= 6.1 Hz, ~0.6H) ppm.

13 C NMR: (125 MHz, Acetone-d6): no multiple conformers observed: δ 173.6 (CO), 169.8 (CO), 161.1 (C), 139.9 (C), 136.5 (C), 134.7 (C), 131.6 (CH), 130.5 (CH), 128.7 (CH), 128.4 (CH), 127.6

(CH), 121.2 (CH), 114.5 (CH), 114.0 (CH), 55.5 (CH3), 50.9 (CH2), 48.9 (CH2), 46.8 (CH), 40.7

(CH2), 26.3 (CH), 23.0 (CH3), 22.5 (CH3) ppm.

241

Part 3: Experimental Procedures

IR (HATR): 3469 (vw), 3057 (w), 2954 (w), 1641 (s), 1600 (s), 1487 (m), 1470 (m), 1434 (m), 1418 (m), 1365 (m), 1342 (m), 1305 (m), 1279 (m), 1258 (m), 1222 (m), 1163 (w), 1037 (w), 1004 (w), 953 (vw), 914 (vw), 873 (w), 779 (m), 761 (m), 747 (m), 725 (m), 693 (m), 678 (w), 634 (w) cm-1.

TLC: Rf = 0.31 (Dichloromethane/MeOH 95/5).

XIII.4.8.5 5-CINNAMYL-3,4-DIHYDRO-3-ISOBUTYLBENZO[g][1,5]DIAZOCINE- 2(1H),6(5H)-DIONE VI.93

F.C.: dichloromethane/MeOH 99/1 O HN HPLC: method B, tret = 30.5

N CYC yield: 44% (yellow glass), 20 mg from 47 mg XIII.5 O

Molecular Formula: C23H26N2O2

Molecular weight: 362.46 g mol-1.

LC-MS: Peak at 6.3 min; ES-MS positive mode [m/z (fragment, intensity)]: 725.3 (2M+H+, 100), 363.3 (M+H+, 25).

+ + HRMS (ESI): Peak for C23H27N2O2 [M+H ]: calculated 363.2072, found 363.2066.

1 H NMR: (500 MHz, Acetone-d6): presence of two inversed boat-type conformers (ratio 70/30, individual assignment not possible): δ 8.75-8.40 (m, NH), 7.60-7.20 (m, 9H), 6.74-6.62 (m, 1H), 6.35-6.24 (m, ~0.7H), 5.80-5.70 (m, ~0.3H), 4.68-4.20 (m, 2H), 3.60-2.50 (m, 3H), 1.66-1.50 (m, 1H), 1.35-1.22 (m, 1H), 1.10-0.98 (m, 1H), 0.90-0.61 (m, 6H) ppm.

13 C NMR: (125 MHz, Acetone-d6): two conformers are present: major conformer: δ 173.8 (CO), 169.2 (CO), 137.9 (C), 136.8 (C), 134.5 (C), 134.1 (CH), 131.5 (CH), 129.8 (CH), 129.5 (CH),

128.7 (CH), 128.5 (CH), 128.4 (CH), 127.6 (CH), 127.5 (CH), 126.0 (CH), 51.5 (CH2), 48.1 (CH2),

47.8 (CH), 40.8 (CH2), 26.4 (CH), 23.0 (CH3), 22.5 (CH3) ppm.

TLC: Rf =0.36 (Dichloromethane/MeOH 95/5).

242

Part 3: Experimental Procedures

XIII.4.8.5 3,4-DIHYDRO-3-(NAPHTH-2-YL)METHYL-5-METHYLBENZO[g][1,5]DIAZOCINE- 2(1H),6(5H)-DIONE VI.94

F.C.: hexane/EtOAc/AcOH 1/1/1 O HN HPLC: method A, tret = 14.9

N CYC yield 80% (colorless glass), 41 mg from 118.2 mg XIII.6 O Molecular Formula: C22H20N2O2

Molecular weight: 344.40 g mol-1.

LC-MS: peak at 5.5 min; ES-MS positive mode [m/z (fragment, intensity)]: 689.2 (2M+H+, 100), 345.1 (M+H+, 64).

+ + HRMS (ESI): Peak for C22H21N2O2 [M+H ]: calculated 345.1603, found 345.1593.

1 H NMR: (400 MHz, Acetone-d6): presence of two inversed boat-type conformers (ratio 75/25, individual assignment not possible): δ 8.70-8.40 (br.m, NH), 7.90-7.60 (m, 4H), 7.50-7.26 (m, 6H), 7.20-7.05 (m, 1H), 3.75-3.40 (m, 2H), 3.35-3.16 (m, 1H), 3.13-3.03 (m, 3H), 3.00-2.80 (m, 2H) ppm.

13 C NMR: (100 MHz, Acetone-d6): no multiple conformers observed: δ 172.5 (CO), 136.5 (C), 136.1 (C), 134.5 (C), 133.4 (C), 131.1 (CH), 128.9 (CH), 128.4 (CH), 128.4 (CH), 128.3 (CH),

128.2 (CH), 128.1 (CH), 127.3 (CH), 126.9 (CH), 126.4 (CH), 52.9 (CH2), 50.1 (CH), 37.8 (CH2),

32.9 (CH3) ppm.

IR (HATR): 3453 (vw), 3049 (w), 2920 (w), 1645 (s), 1632 (s), 1602 (s), 1575 (m), 1505 (w), 1478 (m), 1441 (m), 1401 (s), 1357 (w), 1326 (w), 1271 (w), 1248 (w), 1228 (w), 1192 (vw), 1125 (vw), 1106 (w), 1070 (w), 1037 (w), 1008 (w), 953 (vw), 915 (vw), 898 (vw), 860 (w), 821 (w), 780 (m), 756 (m), 676 (w), 640 (w), 620 (w) cm-1.

TLC: Rf = 0.17 (Hexane/EtOAc/AcOH 40/60/10).

243

Part 3: Experimental Procedures

XIII.4.8.6 8-BROMO-3,4-DIHYDRO-5-ETHYL-3-(NAPHTH-2- YL)METHYLBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VI.95

F.C.: hexane/EtOAc/AcOH 60/40/10 O

HN HPLC: method E, tret = 13.0

Br N CYC yield: 63% (brown glass), 48 mg from 80 mg XIII.7 O Molecular Formula: C23H21BrN2O2

Molecular weight: 437.32 g mol-1.

LC-MS: Peak at 6.2 min; ES-MS positive mode [m/z (fragment, intensity)]: 879 (2M(81Br)+H+, 10), 875.0 (2M(79Br)+H+, 75), 440.0 (M (81Br)+H+, 25), 439.0 (M(81Br), 100), 438.0 (M (79Br)+H+, 25).

79 + + HRMS (ESI): Peak for C23H22 BrN2O2 [M+H ]: calculated 437.0864, found 437.0876.

1 H NMR: (400 MHz, Acetone-d6): presence of two inversed boat-type conformers (ratio ~ 80/20, individual assignment not possible): δ 8.63 and 8.53 (2x br.s, NH, ratio 3/7), 7.90-7.05 (m, 9H), 6.97 (d, J=8.2 Hz, 1H), 3.79-3.35 (m, 5H), 3.25-2.90 (m, 2H), 1.17 (t, J= 7.2 Hz, ~2.8H), 1.11 (t, J= 6.9 Hz, ~0.2H) ppm.

13 C NMR: (100 MHz, Acetone-d6): no multiple conformers observed: δ 172.7 (CO), 169.6 (CO), 138.3 (C), 136.5 (C), 134.6 (C), 134.0 (CH), 133.7 (C), 133.5 (C), 131.3 (CH), 129.8 (CH), 129.3 (CH), 129.0 (CH), 128.5 (CH), 128.4 (CH), 127.0 (CH), 126.5 (CH), 126.3 (CH), 120.7 (C), 51.3

(CH2), 51.1 (CH), 41.5 (CH2), 37.8 (CH2), 13.4 (CH3) ppm.

IR (HATR): 3401 (vw), 3187 (w), 3048 (w), 2975 (w), 1641 (s), 1631 (s), 1506 (w), 1470 (m), 1433 (m), 1383 (m), 1349 (m), 1312 (m), 1279 (m), 1248 (w), 1219 (w), 1189 (w), 1156 (vw), 1122 (w), 1078 (w), 1016 (w), 991 (w), 953 (vw), 899 (w), 863 (w), 818 (m), 744 (m), 697 (vw), 647 (w), 619 (vw) cm-1.

TLC: Rf = 0.10 (Hexane/EtOAc/AcOH 60/40/10).

244

Part 3: Experimental Procedures

XIII.4.8.7 5-(BUT-1-YN-4-YLE)-3,4-DIHYDRO-8-METHOXY-3-(NAPHTH-2- YL)METHYLBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VI.96

F.C.: hexane/EtOAc/AcOH 60/40/10 H O N HPLC: method D, tret = 10.4 MeO N CYC yield: 21% (brown glass), 23 mg from 74 mg XIII.8 O

Molecular Formula: C26H24N2O3

Molecular weight: 412.48 g mol-1.

LC-MS: Peak at 6.0 min, ES-MS positive mode [m/z (fragment, intensity)]: 825.3 (2M+H+, 100), 413.0 (M+H+, 90).

+ + HRMS (ESI): Peak for C26H25N2O3 [M+H ]: calculated 413.1865, found 413.1861.

1 H NMR: (500 MHz, CDCl3): presence of two inversed boat-type conformers (ratio 70/30, individual assignment not possible): δ 7.81-7.38 (m, 6H), 7.24-6.68 (m, 5H), 3.95-3.81 (m, 1H), 3.80 (s, 3H), 3.70-3.25 (m, 4H), 3.30-2.78 (m, 2H), 2.67-2.42 (m, 2H), 1.95 (t, J= 2.5Hz, ~0.3H) 1.87 (t, J = 2.5 Hz, ~0.7H) ppm.

13 C NMR: (125 MHz, CDCl3): two conformers are present: major conformer: δ 172.9 (CO), 168.7 (CO), 159.1 (C), 135.6 (C), 134.4 (C), 133.4 (C), 132.4 (C), 128.5 (CH), 127.8 (CH), 127.6 (CH), 127.4 (CH), 126.9 (CH), 126.3 (CH), 125.8 (CH), 124.7 (C), 116.7 (CH), 112.3 (CH), 80.1 (C),

70.1 (CH), 55.7 (CH3), 51.3 (CH2), 48.8 (CH), 45.1 (CH2), 37.2 (CH2), 17.8 (CH2) ppm.

IR (HATR): 3434 (vw), 3054 (w), 2964 (w), 1637 (s), 1493 (m), 1476 (m), 1432 (m), 1397 (m), 1356 (m), 1317 (m), 1289 (m), 1218 (m), 1180 (w), 1146 (w), 1124 (w), 1108 (w), 1076 (w), 1031 (m), 963 (vw), 909 (w), 860 (w), 817 (m), 727 (m), 643 (m), 622 (m) cm-1.

TLC: Rf = 0.12 (Hexane/EtOAc/AcOH 60/40/10).

245

Part 3: Experimental Procedures

XIII.4.8.8 3,4-DIHYDRO-5-(4-FLUOROBENZYL)-8-METHYL-3-(NAPHTH-2- YL)METHYLBENZO[g][1,5]DIZAOCINE-2(1H),6(5H)-DIONE VI.97

O F.C.: dichloromethane/MeOH 99/1 HN HPLC: method D, tret = 16.6

Me N CYC yield: 41% (colorless glass), 29 mg from 80 mg XIII.9 O

Molecular Formula: C29H25FN2O2 F Molecular weight: 452.51 g mol-1.

LC-MS: Peak at 6.7 min; ES-MS positive mode [m/z (fragment, intensity)]: 905.7 (2M+1H+, 70), + 493.9 (M+CH3CN+H , 10), 452.8 (M, 100).

+ + HRMS (ESI): Peak for C29H26FN2O2 [M+H ]: calculated 453.1978, found 453.1983.

1 H NMR: (400 MHz, CDCl3): presence of two inversed boat-type conformers (ratio ~ 75/25, individual assignment not possible): δ 7.84-6.70 (m, 15H), 5.31 (d, J=14 Hz, ~0.25H), 5.13 (d, J=14.7 Hz, ~0.75H), 4.24 (d, J=14.7 Hz, ~0.75H), 4.15 (d, J=14.9 Hz, ~0.25H), 3.43-3.25 (m, 2H), 3.10-2.90 (m, 2H), 2.83-2.65 (m, 1H), 2.37 and 2.35 (2x br.s, 3H) ppm.

13 C NMR: (100 MHz, CDCl3): two conformers are present: major conformer: δ 172.7 (CO), 169.2 (CO), 162.4 (d, J=246.5 Hz, C), 138.4 (C), 134.4 (C), 133.9 (C), 133.4 (C), 132.4 (C), 132.1 (d, J=2.9 Hz, C), 131.6 (CH), 130.1 (d, J= 8.1 Hz, CH), 129.7 (C), 128.5 (d, J= 7.3 Hz, CH), 127.6 (CH), 127.4 (CH), 126.8 (CH), 126.2 (d, J= 16.1 Hz, CH), 125.8 (CH), 115.6 (d, J= 21.3 Hz, CH),

49.0 (CH2), 48.4 (CH), 47.9 (CH2), 37.2 (CH2), 20.9 (CH3) ppm.

IR (HATR): 3405 (vw), 3194 (w), 3048 (w), 2923 (w), 1637 (s), 1507 (m), 1473 (m), 1437 (m), 1413 (m), 1388 (m), 1352 (m), 1312 (m), 1248 (w), 1219 (m), 1155 (m), 1094 (w), 1033 (w), 1016 (vw), 954 (vw), 925 (vw), 891 (vw), 853 (w), 817 (s), 790 (m), 748 (m), 655 (vw), 627 (vw) cm-1.

TLC: Rf = 0.07 (Dichloromethane/MeOH 99/1).

246

Part 3: Experimental Procedures

XIII.4.8.9 3-(4-CHLOROBENZYL)-3,4-DIHYDRO-8-METHOXY-5- METHYLBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VI.98

Cl F.C.: hexane/EtOAc/AcOH 80/20/10

HPLC: method A, tret = 16.8 O HN CYC yield: 25% (yellow glass), 13 mg from 43mg XIII.10

Molecular Formula: C H ClN O MeO N 19 19 2 3 O Molecular weight: 358.81 g mol-1.

LC-MS: Peak at 5.4 min; ES-MS positive mode [m/z (fragment, intensity)]: 721.2 (2M(35Cl)+H+, 35 + 37 37 + 15), 717.1 (2M( Cl)+H ,90), 400.1 (M( Cl)+CH3CN, 15), 361.0 (M ( Cl)+H , 35), 359.0 (M (35Cl)+H+, 100).

35 + + HRMS (ESI): Peak for C19H20 ClN2O3 [M+H ]: calculated 359.1162, found 359.1146.

1 H NMR: (400 MHz, Acetone-d6): presence of two inversed boat-type conformers (ratio 70/30, individual assignment not possible): δ 8.19 and 8.12 (2x br.s, NH, ratio 3/7), 7.17-6.93 (m, 5H), 6.90-6.83 (m, 1H), 6.80-6.71 (m, 1H), 3.72 and 3.3.71 (2x s, 3H), 3.60-3.02 (m, 3H), 2.92 (s, 3H), 2.64-2.52 (m, 2H) ppm.

13 C NMR: (100 MHz, Acetone-d6): no multiple conformers observed: δ 178.6 (CO), 169.6 (CO), 159.7 (C), 138.1 (C), 132.1 (C), 131.7 (CH), 130.8 (C), 129.3 (CH), 129.1 (CH), 128.0 (C), 116.8

(CH), 112.9 (CH), 56.1 (CH3), 52.9 (CH2), 50.0 (CH), 36.8 (CH2), 33.0 (CH3) ppm.

IR (HATR): 3399 (br. m), 3059 (vw), 2921 (s), 2851 (m), 1659 (m), 1650 (m), 1641 (m), 1632 (m), 1547 (vw), 1492 (m), 1441 (m), 1429 (s), 1410 (s), 1317 (s), 1259 (m), 1232 (m), 1159 (m), 1091 (s), 1058 (s), 1033 (s), 1013 (s), 896 (vw), 872 (w), 820 (m), 799 (m), 767 (w), 718 (w), 703 (vw), 661 (w), 610 (w) cm-1.

TLC: Rf = 0.08 (Hexane/EtOAc/AcOH 60/40/10).

247

Part 3: Experimental Procedures

XIII.4.8.10 3-(4-CHLOROBENZYL)-3,4-DIHYDRO-5- ISOPENTYLBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VI.99

Cl F.C.: hexane/EtOAc/AcOH 80/20/10

Chemical Formula: C22H25ClN2O2 O HN Molecular Weight: 384.89 g mol-1.

LC-MS: Peak at 6.4 min; ES-MS positive mode [m/z (fragment, N O intensity)]: 773.3 (2M(37Cl)/(37Cl), 20), 769.3 (2M(35Cl)/(35Cl), 100), 388.2 (M(37Cl)/(37Cl), 20), 385.2 (M (35Cl)+H+, 65).

35 + + HRMS (ESI): Peak for C22H26 ClN2O2 [M+H ]: calculated 385.1682, found 385.1689.

1 H NMR: (300 MHz, CDCl3): presence of two inversed boat-type conformers (ratio cannot be determined, individual assignment is not possible): δ 7.75 (br.s, NH), 7.55-7.30 (m, 3H), 7.24-6.64 (m, 5H), 3.75-3.20 (m, 4H), 3.15-2.55 (m, 3H), 1.70-1.35 (m, 3H), 1.05-0.65 (m, 6H) ppm.

13 C NMR: (75 MHz, CDCl3): two conformers are present: major conformer δ 172.6 (CO), 168.4 (CO), 135.4 (C), 134.8 (C), 132.8 (C), 132.1 (C), 130.8 (CH), 130.1 (CH), 128.8 (CH), 128.2 (CH),

128.0 (CH), 126.0 (CH), 50.4 (CH2), 49.3 (CH), 44.8 (CH2), 36.5 (CH2), 36.4 (CH2), 26.2 (CH),

22.4 (CH3) ppm.

IR (HATR): 3295 (w), 3055 (w), 2955 (m), 2923 (m), 1637 (s), 1604 (m), 1575 (m), 1488 (m), 1473 (m), 1443 (m), 1420 (m), 1395 (m), 1365 (m), 1317 (m), 1282 (w), 1248 (w), 1230 (w), 1196 (w), 1153 (w), 1116 (w), 1091 (m), 1013 (m), 950 (vw), 914 (vw), 895 (w), 869 (w), 842 (m), 812 (w), 779 (m), 759 (m), 722 (m), 660 (w), 634 (w), 568 (w) cm-1.

TLC: Rf = 0.53 (Dichloromethane/MeOH 95/5).

248

Part 3: Experimental Procedures

XIII.4.8.11 8-BROMO-3-(4-CHLOROBENZYL)-3,4-DIHYDRO-5-(2-(2,5-DIOXOPYRROLIDIN- 1-YL)ETHYL)BENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VI.100

F.C.: hexane/EtOAc/AcOH 80/20/10 Cl

Chemical Formula: C23H21BrClN3O4 O -1 HN Molecular Weight: 518.78 g mol .

LC-MS: Peak at 5.7 min, ES-MS positive mode [m/z (fragment, Br N 81 37 79 37 + O intensity)]: 522.0 (M ( Br, Cl), 27), 521.0 (M ( Br, Cl)+H , O N 24), 520.0 (M (81Br, 35Cl), 100), 519.9 (M(79Br, 35Cl)+H+, 18), O 518.0 (M(79Br, 35Cl), 80).

79 35 + + HRMS (ESI): Peak for C23H22 Br ClN3O4 [M+H ]: calculated 518.0482, found 518.0476.

1 H NMR: (300 MHz, CDCl3): presence of two inversed boat-type conformers (ratio cannot be determined, individual assignment not possible): δ 7.65-7.30 (m, 3H), 7.22 (d, J=8.3 Hz, 2H), 7.10-6.90 (m, 2H), 6.77 (d, J=8.5 Hz, 1H), 4.35-4.05 (m, 1H), 3.95-3.75 (m, 1H), 3.68-3.57 (m, 1H), 3.40-2.93 (m, 4H), 2.88-2.55 (m, 6H) ppm.

13 C NMR: (75 MHz, CDCl3): two conformers are present: major conformer δ 178.2 (CO), 178.0 (CO), 172.9 (CO), 172.4 (CO), 168.6 (C), 135.4 (CH), 134.5 (C), 133.5 (CH), 131.5 (C), 131.3

(CH), 130.6 (C), 129.3 (C), 129.1 (C), 128.9 (C), 128.0 (C), 122.2 (CH), 51.3 (CH2), 50.1 (CH2),

48.9 (CH), 48.1 (CH2), 44.1 (CH2), 36.7 (CH2), 28.7 (CH2) ppm.

IR (HATR): 3493 (vw), 1772 (w), 1697 (s), 1643 (s), 1594 (w), 1472 (m), 1428 (m), 1400 (m), 1354 (m), 1330 (m), 1298 (w), 1251 (w), 1227 (w), 1153 (s), 1119 (m), 1089 (w), 1014 (w), 941 (w), 911 (w), 882 (w), 816 (m), 795 (w), 771 (w), 729 (w), 700 (w), 665 (m), 613 (w), 596 (w) cm- 1 .

TLC: Rf= 0.36 (Dichloromethane/MeOH 95/5).

249

Part 3: Experimental Procedures

XIII.4.8.12 5-BENZYL-8-BROMO-3-(4-CHLOROBENZYL)-3,4- DIHYDROBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VI.101

Cl F.C.: hexane/EtOAc/AcOH 80/20/10

HPLC: method A, tret = 18.2 O HN CYC yield: 27% (yellow glass), 22 mg from 70 mg XIII.11

Br N Molecular Formula: C24H20BrClN2O2 O Molecular weight: 483.78 g mol-1.

LC-MS: Peak at 6.7 min; ES-MS positive mode [m/z (fragment, intensity)]: 971.0 (2M(79Br, 37 + 79 35 + 79 37 + Cl)+H , 30), 967.0 (2M( Br, Cl)+H , 100), 527.1 (M ( Br, Cl)+CH3CN+H , 10), 488.0 (M (81Br, 37Cl)+ H+, 5), 486.0 (M (79Br, 37Cl)+ H+, 35), 485.0 (M (81Br, 35Cl), 90) .

79 35 + + HRMS (ESI): Peak for C24H21 Br ClN2O2 [M+H ]: calculated 483.0474, found 483.0472.

1 H NMR: (400 MHz, CDCl3): presence of two inversed boat-type conformers (ratio cannot be determined, individual assignment not possible): δ 9.2 (br.s, 1H), 7.71-7.49 (m, 2H), 7.41-7.16 (m, 7H), 7.06-6.78 (m, 3H), 5.34 (d, J=14.8 Hz, 0.3H), 5.23 (d, J =14.6 Hz, 0.7H), 4.26 (d, J = 14.8 Hz, 0.7H), 4.14 (d, J = 15.1 Hz, 0.3H), 3.45-2.45 (m, 5H) ppm.

13 C NMR: (100 MHz, CDCl3): two conformers are present: major conformer: δ 172.0 (CO), 167.3 (CO), 135.8 (C), 135.8 (C), 135.0 (C), 134.0 (CH), 133.0 (C), 132.5 (C), 131.2 (CH), 130.2 (CH),

128.9 (CH), 128.9 (CH), 128.4 (CH), 128.1 (CH), 127.6 (CH), 121.8 (C), 49.1 (CH2), 48.6 (CH2),

48.3 (CH), 36.2 (CH2) ppm.

IR (HATR): 3259 (vw), 3059 (w), 3030 (w), 2965 (w), 2916 (w), 1643 (s), 1491 (m), 1470 (m), 1441 (m), 1397 (m), 1355 (m), 1329 (w), 1261 (w), 1202 (w), 1182 (w), 1152 (w), 1091 (m), 1077 (m), 1014 (m), 957 (w), 935 (w), 910 (w), 885 (vw), 812 (m), 799 (m), 762 (w), 731 (m), 698 (m), 664 (w), 639 (vw), 634 (vw), 617 (w) cm-1.

TLC: Rf = 0.47 (Hexane/EtOAc/AcOH 60/40/10).

250

Part 3: Experimental Procedures

XIII.5 SYNTHESIS OF N-FMOC-3-AMINO-3-ALKYLPROPIONIC ACIDS

XIII.5.1 General procedure

O R O R 2 eq Na2CO3, 1 eq Fmoc-OSu, HO NHFmoc HO NH2 THF/H2O 2/1, r.t, o.n VII.8 VII.5

To a solution of β-amino acid VII.8 (11.2 mmol, 1 eq) in a mixture of THF/H2O (112 ml) is added

Na2CO3 (22.3 mmol, 2 eq). After 5 min, Fmoc-OSu (11.2 mmol, 1 eq) is added and the resulting mixture is stirred overnight at room temperature. The reaction mixture is acidified using 6 M HCl until pH=1-2 and after adding (20 ml) H2O, the aqueous phase is extracted three times with ethyl acetate. The combined organic layers are dried with anhydrous magnesium sulfate, the drying agent filtered and the volatiles removed under reduced pressure. The crude solid is purified by flash chromatography (F.C.).

XIII.5.2 3-(N-(9-FLUORENYLMETHYLOXYCARBONYL)AMINO)BUTANOIC ACID VII.9

O F.C.: Dichloromethane/MeOH 98/2 to 85/15 HO NH Yield: 83%; white fluffy powder, 13 g from 5 g XIII.12 O O Molecular Formula: C19H19NO4

Molecular weight: 325.34 g mol-1

LC-MS: Peak at 5.0 min; ES-MS positive mode [m/z (fragment, intensity)]: 326.0 (M+H+, 100).

+ + HR-MS (ESI): C19H20NO4 [M+H ]: calculated 326.1392, found 326.1383.

1 H NMR: (300 MHz, DMSO-d6): presence of two carbamate rotamers on NMR-time scale (ratio cannot be determined): δ 7.89 (d, J = 7.5 Hz, 2H), 7.68 (d, J = 7.4 Hz, 2H), 7.51-7.20 (m , 4H), 4.45-4.15 (m, 3H), 3.94-3.75 (m, 1H), 2.43 (dd, J= 7.0/15.6 Hz, 1H), 2.26 (dd, J= 7.2/15.1 Hz, 1H), 1.08 (d, J= 6.4 Hz, 3H) ppm.

251

Part 3: Experimental Procedures

IR (HATR): 3317 (m), 3046 (w), 2922 (w), 1686 (s), 1532 (s), 1476 (w), 1448 (m), 1411 (w), 1374 (w), 1349 (w), 1272 (m), 1256 (s), 1214 (s), 1164 (w), 1094 (m), 1052 (m), 994 (w), 938 (w), 925 (w), 830 (w), 794 (w), 776 (w), 758 (m), 739 (s), 683 (w), 646 (w), 650 (w), 621 (w) cm-1.

TLC: Rf = 0.44 (Dichloromethane/MeOH 90/10).

XIII.5.3 3-(N-(9-FLUORENYLMETHYLOXYCARBONYL)AMINO)-4-PHENYLBUTANOIC ACID VII.11

F.C.: Dichloromethane/MeOH 99/1 to 97/3

O Yield: 90%; white fluffy powder, 4 g from 2 g XIII.13

HO NH Molecular Formula: C25H23NO4 O O Molecular weight: 401.45g mol-1

LC-MS: Peak at 5.6 min; ES-MS positive mode [m/z (fragment, intensity)]: 402.1 (M+H+, 100).

1 H NMR: (400 MHz, CDCl3): presence of two carbamate rotamers on NMR-time scale (ratio ~ 90/10): δ 6.85 (d, J= 7.4 Hz, 2H), 7.64 (d, J = 7.4 Hz, 2H), 7.40 (dd, J=7.5 Hz, 2H), 7.32-7.10 (m, 3H), 3.94-3.75 (m, 7H), 6.51 and 5.99 (2x br.s, NH, 9/1 ratio), 4.40-4.05 (m, 4H), 2.93 (d, J= 6.9 Hz, 2H), 2.58 (d, J= 6.6 Hz, 1.81H), 2.44 (br.s, 0.22H) ppm. 13 C NMR: (100 MHz, CDCl3): two carbamate rotamers are present: major rotamer δ 172.8 (C), 156.4 (C), 145.2 (C), 142.2 (C), 139.6 (C), 130.3 (CH), 129.2 (CH), 128.5 (CH), 128.0 (CH), 127.2

(CH), 126.2 (CH), 120.8 (CH), 66.8 (CH2), 50.9 (CH), 49.2 (CH), 41.0 (CH2), 39.0 (CH2), ppm. IR (HATR): 3312 (m), 3064 (w), 3031 (w), 2948 (w), 2920 (w), 1668.4 (s), 1620 (m), 1539 (m), 1497 (m), 1475 (w), 1433 (m), 1416 (m), 1317 (m), 1298 (m), 1270 (m), 1214 (s), 1134 (m), 1099 (m), 1081 (m), 1040 (m), 997 (w), 974 (w), 919 (w), 864 (w), 842 (vw), 816 (w), 780 (w), 758 (w), 733 (m), 718 (w), 699 (m), 656 (m), 620 (w) cm-1.

TLC: Rf = 0.46 (Dichloromethane/MeOH 90/10).

252

Part 3: Experimental Procedures

XIII.5.4 3-(N-(9-FLUORENYLMETHYLOXYCARBONYL)AMINO)-5-METHYLHEXANOIC ACID VII.10

F.C.: Dichloromethane/MeOH 99/1 to 96/4

O Yield: 86%; white fluffy powder, 3.4 g from 2 g XIII.14 HO NH Molecular Formula: C22H25NO4 O O Molecular weight: 367.44 g mol-1

LC-MS: Peak at 4.5 min; ES-MS positive mode [m/z (fragment, intensity)]: 368.2 (M+H+, 100).

+ + HR-MS (ESI): C22H26NO4 [M+H ]: calculated 328.1861, found 328.1858.

1 H NMR: (300 MHz, Acetone-d6): presence of two carbamate rotamers on NMR-time scale (ratio 95/5): δ 7.85 (d, J=7.5 Hz, 2H), 7.80-7.63 (m, 1.95H), 7.62-7.54 (m, 0.05H), 7.46-7.05 (m, 4H), 6.36 and 5.83 (2x br.s, NH, ratio 95/5). 4.33 (d, J=6.8 Hz, 1.84 H), 4.48-4.83 (m, 0.16H), 4.27- 4.18 (m, 1H), 4.15-3.99 (m, 1H), 2.59 (d, J=6.4 Hz, 0.22H), 2.54 (d, J= 6.4 Hz, 0.78H), 2.50 (d, J=6.7 Hz, 0.78H), 2.45 (d, J=6.6 Hz, 0.22H), 1.79-1.09 (m, 3H), 0.92 (dd, J= 5.5 Hz, 5.91H), 0.71 (dd, J= 5.3 Hz, 0.09H) ppm. 13 C NMR: (75 MHz, Acetone-d6): two carbamate rotamers are present on NMR-time scale: major rotamer δ 173.3 (CO), 157.1 (C), 145.7 (C), 142.6 (C), 129 (CH), 128.4 (CH), 129.4 (CH), 126.6

(CH), 121.2 (CH), 67.1 (CH2), 48.7 (CH), 47.9 (CH), 45.1 (CH2), 41.2 (CH2), 26.0 (CH), 24.1

(CH3), 22.6 (CH3) ppm. IR (HATR): 3331 (w), 3054 (w), 2957 (w), 1688 (s), 1530 (vw), 1532 (m), 1465 (w), 1447 (m), 1420 (w), 1388 (w), 1365 (w), 1349 (w), 1297 (m), 1262 (m), 1231 (m), 1196 (m), 1155 (w), 1115 (m), 1085 (m), 1039 (m), 994 (w), 972 (w), 936 (w), 851 (w), 799 (vw), 777 (w), 758 (m), 736 (s), 683 (w), 645 (w), 640 (w), 622 (w) cm-1.

TLC: Rf = 0.41 (Dichloromethane/MeOH 90/10).

253

Part 3: Experimental Procedures

XIII.6 SYNTHESIS OF N-CBZ-4-AMINOBUTAN-1-OL

1 eq Et3N, 1 eq PhCH2OCOCl, HO HO NHCbz NH2 0.1 M CH2Cl2, 0C to r.t., 6 h VII.27 VII.22

To a solution of 4-aminobutan-1-ol VII.27 (11.2 mmol, 1 eq) in CH2Cl2 (15 ml) is added Et3N (12.3 mmol, 1.1 eq) and benzyl chloroformate (12.3 mmol, 1.1 eq) at 0 °C. The resulting mixture is stirred for 6 hours at room temperature. The reaction mixture is diluted with saturated NH4Cl (50ml) at 0C. The organic layer washed with brine (50 ml). The combined organic layers are dried with anhydrous magnesium sulfate, the drying agent filtered and the volatiles removed under reduced pressure. The crude liquid is purified by flash chromatography (F.C.).

XIII.6.1 4-N-(CARBOXYBENZYL)BUTAN-1-OL VII.22

F.C.: Dichloromethane/MeOH 99/1 to 96/4 O HO N O Yield: 98%; white powder, 2.4 g from 1 g VII.27 H

Molecular Formula: C12H17NO3

Molecular weight: 223.27 g mol-1

LC-MS: Peak at 4.8 min; ES-MS positive mode [m/z (fragment, intensity)]: 224.2 (M+H+, 100).

+ + HR-MS (ESI): C12H18NO3 [M+H ]: calculated 224.1286, found 224.1277.

1 H NMR: (300 MHz, CDCl3): δ 7.39-7.28 (m, 5H), 5.11 (br.s, 2 H), 3.66 (t, J= 5.8 Hz, 2H), 3.23 (t, J=6.6 Hz, 2H), 1.70-1.52 (m, 4H) ppm. 13 C NMR: (75 MHz, Acetone-d6): δ 156.5 (CO), 136.6 (C), 129.5 (CH), 128.1 (CH), 128.1 (CH),

66.6 (CH2), 62.3 (CH2), 40.9 (CH2), 29.6 (CH2), 26.5 (CH2) ppm. IR (HATR): 3366 (m), 3324 (m), 3040 (w), 2948 (w), 1682 (m), 1586 (w), 1529 (m), 1488 (w), 1365 (w), 1336 (m), 1267 (m), 1233 (m), 1137 (w), 1103 (w), 1054 (m), 1030 (w), 1010 (m), 961 (w), 908 (w), 843 (w), 781 (w), 749 (w), 727 (m), 696 (w), 638 (w) cm-1.

TLC: Rf = 0.2 (Dichloromethane/MeOH 90/10).

254

Part 3: Experimental Procedures

XIII.7 SYNTHESIS OF 4-SUBSTITUTED-1,5-BENZODIAZOCINE-2,6-DIONES

XIII.7.1 β3-Amino acid coupling on Wang resin

1. 2 eq Fmoc-AA-OH, 2 eq DIC, 0.2 eq DMAP, CH Cl , r.t, 24 h 2 2 O R1 2. Ac2O/DIPEA/CH2Cl2 (1/1/3), r.t, 2x1 h OH O NH2 3. 20% 4-methylpiperidine in DMF, 2x20 min VII.6 VII.16

General procedure: Preactivation: To a cooled (0°C) solution of N-Fmoc-β3-amino acid VII.9-

VII.11 (2 eq) in CH2Cl2 (5 ml), DIC (5.449 mmol, 2 eq) is added. The reaction mixture is stirred for 20 minutes at 0 °C.

Coupling: The crude preactivation mixture is transferred to a solid-phase reaction vessel containing Wang resin (0.5 mmol, 1 eq, calculated from manufacturer’s loading 1.9 mmol/g), washed and preswollen with CH2Cl2) after which DMAP is added (0.5449 mmol, 0.2 eq). The suspension is shaken for 24 hours at room temperature after which the resin is filtered and washed consecutively with CH2Cl2 (3x), DMF (3x), MeOH (3x), and CH2Cl2 (3x).

Capping: The resin is suspended in Ac2O/DIPEA/CH2Cl2 (1/1/3, 10 ml) and shaken for 1 h. The resin is filtered and washed with CH2Cl2. This capping procedure is repeated once. The resin is filtered and consecutively washed with CH2Cl2 (3x), DMF (3x), MeOH (3x) and CH2Cl2 (3x) and dried under reduced pressure.

Loading: The loading of the resulting resins was determined by Fmoc UV quantification: Fmoc- β-Homoala-Wang: 0.6690 mmol/g, Fmoc-β-Homoleu-Wang: 0.6894 mmol/g, Fmoc-Homophe- Wang: 0.6429 mmol/g.Yields of final products were calculated in reference to these loading values. An amount of resin-bound Fmoc-amino acid is suspended in a 20% 4-methylpiperidine solution in DMF. The absorbance of the dibenzofulvene/4-methylpiperidine adduct at 300 nm is correlated to its concentration using a calibration line.

Deprotection: The resin is washed with DMF (3x) and treated with a solution of 20% 4- methylpiperidine in DMF for 20 min. The resin is filtered and washed with DMF (3x). The 4- methylpiperidine treatment is repeated once. The resin is subsequently filtered and washed with

DMF (3x), MeOH (3x), CH2Cl2 (3x) and DMF (3x).

255

Part 3: Experimental Procedures

-Amount of resin. For synthesis of the individual library members we used 0.2427 mmol of the above-prepared Fmoc-β-amino acid-loaded Wang resins.

XIII.7.2 Nosyl coupling

O R1 O R1 O O NO2 5 eq o-Ns-Cl, 10 eq collidine, S O NHFmoc O N CH2Cl2, r.t, 2 x 1 h H VII.16 VII.17

General procedure: to a suspension of resin VII.16 (0.428 g, 0.243 mmol, 1 eq) in dry DCE (6 ml) is added 2,4,6-collidine (321 μl, 2.427 mmol, 10 eq) and 2-nitrobenzenesulfonyl chloride (269 mg, 1.213 mmol, 5 eq). After 1 hour shaking, the resin is filtered and washed with DMF (3x15 ml), MeOH (3x15 ml) and CH2Cl2 (3x15 ml). This procedure is repeated once, delivering the nosyl protected resin bound amino acid VII.17.

This reaction was optimized for compounds VII.16, with R1=methyl or benzyl. LC-MS analysis after cleavage of a small amount of resin-bound material confirmed the identity of the expected products.

Molecular Formula: C10H12N2O6S O O O NO2 S -1 HO N Molecular Weight: 288.27 g mol . H LC-MS: Peak at 3.5 min; ES-MS negative mode [m/z (fragment, intensity)]: 287.0 (M-H+, 100).

Molecular Formula: C16H16N2O6S

Molecular Weight: 364.37 g mol-1. O O O NO2 S HO N LC-MS: Peak at 4.5 min; ES-MS positive mode [m/z (fragment, H + + intensity)]: 382.0 (M+ NH4 , 100), 365.0 (M+H , 18).

256

Part 3: Experimental Procedures

XIII.7.3 Mitsunobu alkylation

O R1 O O NO2 O R1 O O NO2 S S O N 10 eq VII.20-VII.26, 5 eq Ph3P, O N H 5 eq DIAD, DCE, r.t, 3x2h R2 VII.17 VII.18

General procedure: the resin was suspended in DCE (9.2 ml) and triphenylphosphine (319 mg, 1.214 mmol, 5. eq), alcohol VII.20-VII.26 (2.427 mmol, 10 eq) and DIAD (318 ml, 1.214 mmol, 5 eq) were sequentially added. The reaction mixture is shaken for 2 h at room temperature after which the resin is drained and washed with dry dichloromethane and dry DCE. This Mitsunobu procedure was repeated twice, the resin was filtered and washed with DMF (3x15 ml), MeOH

(3x15 ml) and CH2Cl2 (3x15 ml).

This reaction was optimized for compounds VII.18, with (R1=methyl, R2=methyl), (R1=methyl,

R2=benzyl), (R1=benzyl, R2=methyl) or (R1=benzyl, R2=benzyl). LC-MS analysis after cleavage of a small amount of resin-bound material confirmed the identity of the expected products.

Molecular Formula: C11H14N2O6S O O O NO2 S HO N Molecular Weight: 302.3 g mol-1.

LC-MS Peak at 3.7 min; ES-MS positive mode [m/z (fragment, + + intensity)]: 320.0 (M+NH4 , 100), 303.1 (M+H , 35).

Molecular Formula: C17H18N2O6S O O O NO2 S -1 HO N Molecular Weight: 378.39 g mol .

LC-MS: Peak at 4.8 min; ES-MS positive mode [m/z (fragment, + + intensity)]: 396.1 (M+NH4 , 58), 379.1 (M+H , 100).

Molecular Formula: C17H18N2O6S

Molecular Weight: 378.39 g mol-1 O O O NO2 S HO N LC-MS: Peak at 4.7 min; ES-MS positive mode [m/z (fragment, + intensity)]: 396.1 (M+H2O, 100), 379.0 (M+H , 20).

257

Part 3: Experimental Procedures

Molecular Formula: C23H22N2O6S

Molecular Weight: 454.49 g mol-1 O O O NO2 S HO N LC-MS: Peak at 5.5 min; ES-MS positive mode [m/z (fragment, + intensity)]: 472.1 (M+H2O, 100), 455.0 (M+H , 100).

XIII.7.4 Nosyl removal

O O O NO2 O S O N 2.5 eq DBU, 5 eq HSCH2CH2OH O NH DMF, r.t, 2x30min R1 Me R1 R2 VII.18 VII.19

General procedure: the resin is suspended in DMF (4.8 ml) and DBU (90.7 μl, 0.6067 mmol, 2.5 eq) and 2-mercaptoethanol (85.1 μl, 1.214 mmol, 5 eq) were added. The resulting suspension was shaken for 30 min after which the resin was drained and washed with DMF (3x15 ml), MeOH

(3x15 ml) and CH2Cl2 (3x15 ml). This procedure is repeated once, after which the resin was drained and washed with DMF (3x15ml).

This reaction was optimized for compounds VII.19, with R1=benzyl, R2=benzyl. LC-MS analysis after cleavage of a small amount of resin-bound material confirmed the identity of the expected products.

Molecular Formula: C17H19NO2

-1 O Molecular Weight: 269.33 g mol .

HO NH LC-MS: Peak at 4.3 min; ES-MS positive mode [m/z (fragment, intensity)]: 270.1 (M+H+, 100).

258

Part 3: Experimental Procedures

XIII.6.5 Coupling of Fmoc-anthranilic acids

O R1 O R1 O NHFmoc 10 eq VI.7-VI.10 and VII.7 , 5 eq DIC O NH O N CH2Cl2/DMF 9/1, r.t, 24 h R2 R2 VII.19 VII.28 R3

General procedure: Preactivation: To a cooled solution (0°C) of the appropriate N-Fmoc- protected anthranilic acid derivative VI.7-VI.10 and VII.7 (2.427 mmol, 10 eq) in CH2Cl2/DMF (9/1) (6 ml), is added DIC (127.6 μl, 1.214 mmol, 5 eq) and the reaction mixture was stirred for 30 min at 0 °C.

Coupling: The crude preactivation mixture was transferred to the appropriate resin VII.19, preswollen in CH2Cl2/DMF 9/1, and the resulting suspension is shaken for 24 h. The resin was drained and washed consecutively with DMF (3x15 ml), MeOH (3x15 ml) and CH2Cl2 (3x15 ml).

This reaction was optimized for compounds VII.28, with R1=benzyl, R2=methyl. LC-MS analysis after cleavage of a small amount of resin-bound material confirmed the identity of the expected products.

Molecular Formula: C33H30N2O5

Molecular Weight: 534.60 g mol-1. O O NHFmoc

HO N LC-MS: Peak at 6.4 min; ES-MS positive mode [m/z (fragment, intensity)]: 535.2 (M+H+, 100).

XIII.7.6 Fmoc removal

O R O NHFmoc 1 O R1 O NH2 O N 20 % 4-methylpiperidine in DMF O N R2 r.t, 2x20min R R 2 VII.28 3 VII.31 R3

Removal of the Fmoc protecting group, see section XIII.6.1

259

Part 3: Experimental Procedures

XIII.7.7 Cleavage from the resin

O R1 O NH2 O R1 O NH2 TFA/H O 9/1, 1 h. O N 2 HO N

R2 R2 R R VII.31 3 VII.32 3

General procedure: resin VII.31 is suspended in a mixture of trifluoroacetic acid/water 95/5 (10 ml) and shaken for 1 hour at room temperature. The resin is filtered and washed extensively with dichloromethane. The filtrate is concentrated under reduced pressure and the obtained residue is further purified by flash chromatography (F.C.) or preparative reversed-phase HPLC.

XIII.7.7.1 3-(N-ANTHRANOYL,N-METHYL)BUTANOIC ACID XIII.15

F.C.: dichloromethane/MeOH 99/1 to 97/3

O O NH2 SPS yield: 77% (colorless foam), 63 mg from 700 mg resin VII.16 HO N

Molecular Formula: C12H16N2O3

Molecular weight: 236.26 g mol-1.

LC-MS: Peak at 3.411 min; ES-MS positive mode [m/z (fragment, intensity)]: 237.1 (M+H+, 100).

+ + HRMS (ESI): Peak for C12H17N2O3 [M+H ] calculated: 237.1239, found: 237.1236.

1 H NMR: (300 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (two rotamers ratio 55/45): δ 7.15 (t, J = 7.6 Hz, 1H), 7.10-6.95 (m, 3H), 6.74 (br.s, 2H), 5.30-4.71 (m, ~0.55H), 4.70-4.03 (m, ~0.45H), 2.85 (br.s, 3H), 2.70-2.22 (m, 2H), 1.26 (d, J =6.6 Hz, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): no multiple conformers observed: δ 171.9 (CO), 130.5 (CH), 127.5

(CH), 127.4 (CH), 127.2 (CH), 118.3 (C), 117.1 (C), 52.1 (CH), 38.04 (CH2), 31.6 (CH3), 18.0

(CH3) ppm.

IR (HATR): 3448 (w), 3360 (w), 2969 (w), 1715 (m), 1608 (s), 1578 (s), 1494 (m), 1483 (m), 1453 (m), 1400 (m), 1356 (m), 1304 (m), 1255 (m), 1197 (m), 1159 (m), 1135 (m), 1064 (m), 1032 (m), 938 (w), 888 (w), 860 (w), 837 (vw), 797 (w), 749 (s), 721 (w), 667 (vw), 640 (w) cm-1.

260

Part 3: Experimental Procedures

TLC: Rf = 0.15 (dichloromethane /MeOH 97/3).

XIII.7.7.2 3-(N-ANTHRANOYL,N-BENZYL)BUTANOIC ACID XIII.16

F.C.: Hexane/EtOAc/AcOH 55/45/1 O O NH2 HO N SPS yield: 46% (colorless glass), 66 mg from 700 mg resin VII.16

Molecular Formula: C18H20N2O3

Molecular weight: 312.36 g mol-1.

LC-MS: Peak at 4.340 min; ES-MS positive mode [m/z (fragment, intensity)]: 313.1 (M+H+, 100).

+ + HRMS (ESI): Peak for C18H21N2O3 [M+H ] calculated: 313.1552, found: 313.1548.

1 H NMR: (400 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio ~50/50): δ 7.50-6.50 (m, 11H), 4.49 (br.s, 2H), 3.80-3.55 (m, ~0.48H), 3.28-2.95 (m, ~0.52H) 2.86-2.29 (m, 2H), 1.22 (d, J = 6.5 Hz, 3H) ppm.

13 C NMR: (100 MHz, CDCl3): two amide rotamers are present on NMR-time scale: major rotamer δ 174.2 (CO), 171.0 (C), 137.6 (C), 131.4 (CH), 128.7 (CH), 127.0 (broad, CH), 124.5 (CH), 60.9

(CH2), 52.7 (CH), 38.2 (CH2), 19.7 (CH3) ppm.

IR (HATR): 3457 (vw), 3338 (vw), 3028 (w), 2938 (w), 2934 (w), 1704 (m), 1665 (m), 1614 (m), 1589 (m), 1495 (m), 1441 (m), 1421 (m), 1383 (w), 1349 (m), 1298 (w), 1186 (s), 1133 (s), 1087 (m), 1078 (m), 1018 (m), 975 (w), 911 (vw), 855 (vw), 836 (w), 797 (w), 781 (w), 749 (m), 721 (m), 698 (m), 632 (w) cm-1.

TLC: Rf = 0.05 (dichloromethane /MeOH 97/3).

261

Part 3: Experimental Procedures

XIII.7.7.3 3-(N-(-2-AMINO-5-METHOXYBENZOYL),N-(3- TRIFLUOROMETHYLBENZYL))BUTANOIC ACID XIII.17

F.C.: dichloromethane /MeOH 98/2 O O NH2 HO N SPS yield 57% (brown glass), 110 mg from 192 mg resin VII.16

Molecular Formula: C H F N O OMe 20 21 3 2 4

-1 CF3 Molecular weight: 410.38 mol.g .

LC-MS: Peak at 4.5 min; ES-MS positive mode [m/z (fragment, intensity)]: 411.1 (M+H+, 100).

+ + HRMS (ESI): Peak for C20H22F3N2O4 [M+H ] calculated: 411.1531, found: 411.1531.

1 H NMR (300 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio 75/25): δ 8.79 (s, 2H), 7.58-7.31 (m, 5H), 6.98-6.55 (m, 2H), 5.00 (d, J =15.2 Hz, 1H), 4.50 (br.s, 1H), 4.28 (d, J =15 Hz, 1H,), 3.81 (br.s, ~2.3H), 3.46 (br.s, ~0.7H), 2.77-2.30 (m, 2H), 1.35-1.18 (m, 3H) ppm.

13 C NMR (100 MHz, CDCl3): no multiple conformers observed: δ 170.5 (CO), 158.8 (C), 138.8 (C), 130.7 (C), 130.5 (CH), 129.2 (CH), 126.5 (CH), 125.8 (C), 124.1 (CH), 123.7 (CH), 122.8

(C), 115.2 (CH), 113.3 (CH), 55.7 (CH3), 52.8 (CH), 43.6 (CH2), 38.2 (CH2), 19.8 (CH3) ppm.

IR (HATR): 3418 (w), 2974 (w), 2936 (w), 2841 (w), 1668 (m), 1632 (m), 1598 (s), 1503 (m), 1455 (m), 1437 (m), 1421 (m), 1326 (s), 1290 (m), 1265 (m), 1191 (s), 1163 (s), 1120 (s), 1096 (m), 1073 (m), 1029 (m), 987 (w), 943 (vw), 914 (w), 877 (w), 831 (w), 797 (m), 751 (w), 721 (m), 701 (m), 656 (w), 632 (vw) cm-1.

TLC: Rf = 0.47 (dichloromethane /MeOH 95/5).

262

Part 3: Experimental Procedures

XIII.7.7.4 3-(N-(-2-AMINO-5-METHOXYBENZOYL),N-(CARBOXYBENZYLAMINOBUT-4- YL))BUTANOIC ACID XIII.18

F.C.: dichloromethane /MeOH 99/1 to 98/2 O O NH2 HO N SPS yield 41% (pale brown glass), 89 mg from 700 mg resin VII.16

Molecular Formula: C24H31N3O6 OMe Molecular weight: 457.51 mol.g-1. O NH O LC-MS: Peak at 4.3 min; ES-MS positive mode [m/z (fragment, intensity)]: 458.2 (M+H+, 100).

+ + HRMS (ESI): Peak for C24H32N3O6 [M+H ] calculated: 458.2291, found: 458.2289.

1 H NMR (300 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 9.08 (br.s, 2H), 7.62-6.69 (m, 8H), 5.07 (br.s 2H), 4.55-4.21 (m, 1H), 3.77 (br.s, 3H), 3.55-2.20 (m, 6H), 1.75-1.07 (m, 7H) ppm.

13 C NMR (100 MHz, CDCl3): no multiple conformers observed: δ 156.7 (C), 136.6 (C), 129.3 (C),

128.5 (CH), 128.0 (CH), 128.0 (CH), 117.8 (C), 115.5 (CH), 112.8 (CH), 66.5 (CH2), 55.6 (CH3),

52.1 (CH), 40.7 (CH2), 40.2 (CH2), 38.4 (CH2), 30.9 (CH3), 27.4 (CH2), 25.6 (CH2) ppm.

IR (HATR): 3333 (w), 2939 (w), 2874 (w), 1673 (s), 1622 (m), 1593 (m), 1503 (s), 1455 (m), 1430 (m), 1382 (m), 1354 (m), 1326 (m), 1288 (m), 1259 (m), 1197 (s), 1181 (s), 1132 (s), 1086 (m), 1028 (m), 912 (w), 879 (w), 833 (w), 798 (m), 776 (m), 737 (m), 720 (m), 697 (m), 640 (w) cm-1.

TLC: Rf = 0.04 (dichloromethane /MeOH 95/5).

XIII.7.7.5 3-(N-ANTHRANOYL,N-METHYL)-4-PHENYLBUTANOIC ACID XIII.19

F.C.: dichloromethane /MeOH 97/3

O O NH2 SPS yield: 76% (yellow glass) 130 mg from 442 mg resin VII.16

HO N Molecular Formula: C18H20N2O3

263

Part 3: Experimental Procedures

Molecular weight: 312.36 mol.g-1.

LC-MS: Peak at 4.2 min; ES-MS positive mode [m/z (fragment, intensity)]: 625.2 (2M+H+, 5), 313.1 (M+H+, 100).

+ + HRMS (ESI): Peak for C18H21N2O3 [M+H ] calculated: 313.1552, found: 313.1558.

1 H NMR (400 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio ~ 55/45): δ 7.58 (br.s, 2H), 7.37-6.80 (m, 7H), 6.77-6.30 (m, 2H), 5.33-5.10 (m, ~0.54H), 4.50-4.15 (m, ~0.46H), 3.10-2.29 (s, 7H) ppm.

13 C NMR (100 MHz, CDCl3): two amide rotamers are present on NMR time scale: major rotamer: δ 174.5 (CO), 171.8 (CO), 141.3 (C), 137.2 (C), 130.5 (CH), 129.2 (CH), 129.0 (CH), 128.6 (CH),

127.2 (CH), 122.3(C), 119.3 (CH), 117.6 (CH), 57.8 (CH), 38.2 (CH2), 36.2 (CH2), 27.3 (CH3) ppm.

IR (HATR): 3455 (vw), 3362 (w), 3059 (w), 3026 (w), 2926 (w), 1708 (s), 1611 (s), 1582 (s), 1494 (m), 1453 (m), 1401 (m), 1309 (m), 1263 (m), 1193 (s), 1135 (s), 1062 (m), 1030 (m), 982 (vw), 965 (w), 909 (w), 832 (w), 737 (w), 749 (s), 731 (s), 722 (s), 699 (s), 642 (m) cm-1.

TLC: Rf = 0.05 (dichloromethane /MeOH 97/3).

XIII.7.7.6 3-(N-ANTHRANOYL,N-BENZYL)-4-PHENYLBUTANOIC ACID XIII.20

F.C.: dichloromethane /MeOH 99/1

HPLC: method A, tret = 12.3 O O NH2 HO N SPS yield 48% (yellow glass), 72 mg from 822 mg resin VII.16

Molecular Formula: C24H24N2O3

Molecular weight: 388.45 mol.g-1

LC-MS: Peak at 5.0 min; ES-MS positive mode [m/z (fragment, intensity)]: 389.2 (M+H+, 100).

+ + HRMS (ESI): Peak for C24H25N2O3 [M+H ] calculated: 389.1865, found: 389.1866.

264

Part 3: Experimental Procedures

1 H NMR (400 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.59-6.61 (m, 14 H), 5.18-4.10 (m, 2H), 3.80-2.25 (m, 5H) ppm.

13 C NMR (100 MHz, CDCl3): two amide rotamers are present on NMR-time scale: major rotamer

δ 138.3 (C), 129.3 (C), 129.1 (C), 128.7 (CH), 128.7 (CH), 128.5 (CH), 126.8 (CH), 54.5 (CH2),

42.9 (CH), 42.5 (CH2) 34.5 (CH2) ppm.

IR (HATR): 3447 (w), 3363 (w), 3060 (w), 3026 (w), 2926 (w), 1713 (m), 1612 (s), 1587 (s), 1494 (m), 1453 (m), 1412 (m), 1365 (m), 1305 (m), 1246 (m), 1216 (m), 1155 (m), 1108 (w), 1077 (w), 1027 (m), 978 (w), 959 (w), 908 (w), 873 (w), 854 (w), 809 (vw), 749 (s), 698 (s), 652 (w), 632 (vw) cm-1.

TLC: Rf = 0.15 (dichloromethane /MeOH 97/3).

XIII.7.7.7 3-(N-(2-AMINO-5-BROMOBENZOYL),N-(PENT-1-YL))-4-PHENYL BUTANOIC ACID XIII.21

F.C.: dichloromethane /MeOH 99/1

SPS yield 44% (beige glass), 101 mg from 800 mg resin VII.16 O O NH2 HO N Molecular Formula: C22H27BrN2O3

Molecular weight: 447.36 mol.g-1. Br LC-MS: Peak at 5.1 min; ES-MS positive mode [m/z (fragment, intensity)]: 450.1 (M(81Br)+H+, 20), 449.0 (M(79Br)+H+, 100), 448.1 (M(81Br), 20), 447.1 (M(79Br), 95).

79 + + HRMS (ESI): Peak for C22H28 BrN2O3 [M+H ] calculated: 447.1283, found: 477.1275.

1 H NMR (400 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.41-6.45 (m, 10H), 4.37 (br.s, 1H), 3.73-2.42 (m, 6H), 1.84-0.77 (m, 9H) ppm.

13 C NMR (100 MHz, CDCl3): no multiple conformers observed: δ 170.4 (CO), 138.4 (C), 132.9 (CH), 129.5 (CH), 129.3 (CH), 129.2 (CH), 128.7 (CH), 128.5 (CH), 127.1 (CH), 126.8 (CH), 60.8

(CH2), 42.9 (CH), 42.5 (CH2), 39.8 (CH2), 36.9 (CH2), 28.5 (CH2), 21.9 (CH2), 13.8 (CH3) ppm.

265

Part 3: Experimental Procedures

IR (HATR): 3446 (w), 3362 (w), 3065 (w), 3026 (w), 2954 (m), 2929 (m), 2869 (w), 1711 (s), 1605 (s), 1580 (s), 1485 (s), 1454 (s), 1401 (m), 1361 (m), 1280 (m), 1255 (m), 1200 (s), 1144 (s), 1104 (m), 1080 (w), 1029 (m), 885 (w), 816 (m), 749 (m), 722 (w), 699 (s), 668 (w), 621 (w) cm- 1.

TLC: Rf = 0.23 (dichloromethane /MeOH 9/1).

XIII.7.7.8 3-(N-(2-AMINO-4-TRIFLUOROMETHYLBENZOYL),N-(2-METHOXYETHYL))-4- PHENYLBUTANOIC ACID XIII.22

F.C.: dichloromethane /MeOH 98/2

HPLC: method A, tret = 12.3 O O NH2 SPS yield 56% (yellow glass), 130 mg from 800 mg resin VII.16 HO N

CF3 Molecular Formula: C21H23F3N2O4 OMe Molecular weight: 424.41 mol.g-1.

LC-MS: Peak at 4.7 min, ES-MS positive mode [m/z (fragment, intensity)]: 425.0 (M+H+, 100).

+ + HRMS (ESI): Peak for C21H24F3N2O4 [M+H ] calculated: 425.1688, found: 425.1697.

1 H NMR (400 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.55-6.90 (m, 8H), 6.8 (br.s, 2H), 4.38 (br.s, 1H), 4.10-2.85 (m, 11H) ppm.

13 C NMR (100 MHz, CDCl3): no multiple conformers observed: δ 171.3 (CO), 132.3 (d, J= 31.5 Hz, C), 130.7 (CH), 129.9 (CH), 129.9 (CH), 127.8 (CH), 125.1 (d, J= 271.5 Hz, C), 113.4 (CH),

112.9 (q, J= 4.4 Hz, CH), 71.1 (CH2), 59.1 (CH3), 43.1 (CH2), 39.3 (CH2), 30.7 (CH2), 30.6 (CH) ppm.

IR (HATR): 3461 (w), 3362 (w), 3059 (w), 3027 (w), 2930 (w), 2891 (w), 1713 (m), 1624 (m), 1594 (m), 1544 (w), 1510 (w), 1495 (w), 1436 (m), 1334 (s), 1287 (w), 1253 (m), 1202 (w), 1163 (m), 1116 (s), 1089 (s), 1030 (w), 1015 (w), 924 (w), 870 (w), 817 (m), 777 (w), 747 (w), 700 (m), 670 (w), 652 (vw) cm-1.

TLC: Rf = 0.5 (dichloromethane /MeOH 95/5).

266

Part 3: Experimental Procedures

XIII.7.7.9 3-(N-(2-AMINO-5-BROMOBENZOYL),N-(4-FLUOROBENZYL))-4- PHENYLBUTANOIC ACID XIII.23

F.C.: dichloromethane /MeOH 98/2

HPLC: method A, tret = 12.8 O O NH2 HO N SPS yield: 56% (colorless glass), 140 mg from 800 mg resin VII.16

Molecular Formula: C24H22BrFN2O3 Br F Molecular weight: 485.35 mol.g-1.

LC-MS: Peak at 5.1 min; ES-MS positive mode [m/z (fragment, intensity)]: 488.0 (M(Br81)+H+, 25), 486.1 (M(Br79)+H+, 25).

79 + + HRMS (ESI): Peak for C24H23 BrFN2O3 [M+H ] calculated: 485.0876, found: 485.0862.

1 H NMR (400 MHz, acetone-d6): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.59-6.45 (m, 12H), 5.85-4.21 (m, 3H), 3.09-2.53 (m, 4H) ppm.

13 C NMR (100 MHz, aceone-d6): two amide rotamers are present on NMR-time scale: individual signals cannot be assigned; all signals are listed: δ 139.7 (C), 133.9 (C), 133.4 (CH), 133.3 (CH), 131.6 (CH), 131.6 (CH), 131.5 (CH), 131.5 (CH), 131.2 (CH), 130.8 (CH), 130.6 (CH), 130.5 (CH), 129.9 (CH), 129.9 (CH), 129.8 (CH), 129.8 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH),

122.3 (CH), 119.1 (CH), 116.6 (CH), 116.4 (CH), 116.1 (CH), 108.5 (C) 59.8 (CH), 44.8 (CH2),

42.7 (CH2), 40.9 (CH2), 38.8 (CH2) ppm.

IR (HATR): 3449 (w), 3365 (w), 3059 (w), 3026 (w), 2932 (w), 1714 (s), 1661 (s), 1608 (s), 1583 (s), 1508 (s), 1486 (m), 1453 (m), 1437 (m), 1413 (m), 1368 (m), 1295 (m), 1221 (s), 1201 (s), 1155 (s), 1142 (s), 1097 (m), 1029 (w), 1014 (w), 985 (w), 961 (w), 890 (w), 817 (m), 749 (m), 721 (w), 700 (m), 661 (w), 620 (w) cm-1.

TLC: Rf = 0.11 (dichloromethane /MeOH 95/5).

267

Part 3: Experimental Procedures

XIII.7.7.10 3-(N-ANTHRANOYL,N-METHYL-)-5-METHYLHEXANOIC ACID XIII.24

F.C.: dichloromethane /MeOH 98/2 to 97/3

O O NH2 SPS yield: 99% (beige glass), 133 mg from 428 mg resin VII.16 HO N Molecular Formula: C15H22N2O3

Molecular weight: 278.34 mol.g-1.

LC-MS: Peak at 4.0 min; ES-MS positive mode [m/z (fragment, intensity)]: 557.2 (2M+H+, 15), 279.1 (M+H+, 100).

+ + HRMS (ESI): Peak for C15H23N2O3 [M+H ] calculated: 279.1708, found: 279.1695.

1 H NMR (400 MHz, CDCl3): presence of two amide rotamers on NMR-time scale (ratio ~ 60/40): δ 8.70-7.01 (m, 4H), 6.8 (br.s, 2H), 5.18 (br.s, ~0.6H), 4.09 (br.s, 0.4H), 2.86 and 2.78 (2x br.s, 3H), 2.65-2.29 (m, 2H), 1.71-1.20 (m, 3H), 1.03-0.60 (m, 6H) ppm.

13 C NMR (100 MHz, CDCl3): two amide rotamers are present on NMR-time scale: major rotamer

δ 174.7 (CO) 130.0 (CH), 127.7 (CH), 125.8 (CH), 123.8 (CH), 49.0 (CH), 41.0 (CH2), 30.8 (CH3),

29.6 (CH2), 24.9 (CH), 23.1 (CH3), 21.9 (CH3) ppm.

IR (HATR): 3452 (w), 3362 (w), 2956 (m), 2928 (w), 2870 (w), 1711 (m), 1671 (m), 1612 (s), 1583 (s), 1496 (m), 1453 (m), 1402 (m), 1368 (m), 1337 (m), 1266 (m), 1239 (m), 1185 (s), 1134 (s), 1062 (m), 1032 (m), 955 (w), 921 (w), 901 (w), 873 (w), 834 (w), 797 (w), 749 (m), 720 (m), 642 (w) cm-1.

TLC: Rf = 0.18 (dichloromethane /MeOH 95/5).

XIII.7.7.11 3-(N-ANTHRANOYL,N-BENZYL)-5-METHYLHEXANOIC ACID XIII.25

F.C.: dichloromethane /MeOH 98/2

O O NH2 SPS yield: 73% (brown glass), 125 mg from 428 mg VII.16 HO N Molecular Formula: C21H26N2O3

Molecular weight: 354.44 mol.g-1.

268

Part 3: Experimental Procedures

LC-MS: Peak at 4.7 min, ES-MS positive mode [m/z (fragment, intensity)]: 355.1 (M+H+, 100).

+ + HRMS (ESI): Peak for C21H27N2O3 [M+H ] calculated: 355.2021, found: 355.2020.

1 H NMR (400 MHz, Acetone-d6): presence of two amide rotamers on NMR-time scale (ratio cannot be determined): δ 7.52-6.50 (m, 11H), 4.85-4.30 (m, 3H), 2.88-2.48 (m, 2H), 1.64-1.42 (m, 2H), 0.95-0.55 (m, 6H) ppm.

13 C NMR (100 MHz, Acetone-d6): two amide rotamers are present on NMR-time scale: individual signals cannot be assigned; all signals are listed: δ 173.5 (CO), 170.7 (CO), 140.8 (C), 138.8 (C), 131.9 (C), 131.2 (CH), 130.8 (CH), 129.6 (CH), 129.5 (CH), 128.9 (CH), 128.7 (CH), 126.7 (CH),

122.9 (CH), 118.1 (CH), 55.7 (CH), 45.2 (CH2), 43.4 (CH2), 38.4 (CH2), 25.4 (CH), 23.2 (CH3),

23.1 (CH3) ppm.

IR (HATR): 3449 (w), 3362 (w), 3061 (w), 3029 (w), 2956 (m), 2868 (w), 1713 (m), 1681 (m), 1614 (s), 1586 (s), 1494 (m), 1449 (m), 1437 (m), 1420 (m), 1366 (m), 1351 (m), 1304 (m), 1281 (m), 1262 (m), 1203 (m), 1181 (s), 1150 (s), 1110 (m), 1077 (w), 1029 (w), 1002 (w), 977 (w), 961 (w), 916 (w), 839 (w), 819 (vw), 798 (w), 782 (w), 751 (m), 735 (m), 722 (m), 698 (m), 673 (vw), 646 (w), 616 (vw) cm-1.

TLC: Rf = 0.23 (DCM/MeOH 95/5).

269

Part 3: Experimental Procedures

XIII.7.8 Cyclization in solution

O O R O NH H 1 2 N

HO N DCC-PS, CH2Cl2, R3 R 0.01M, 1h, r.t 2 N R1 R3 VII.32 O R2 VII.1

General procedure: To a solution of purified α,ω-aminocarboxylic acids (0.048 mmol, 1eq) in

CH2Cl2 (4.8 ml) is added polystyrene-bound DCC (loading 2.3 mmol/g, 0.141 mmol, 2.9 eq). The obtained suspension is shaken for 1 h at room temperature after which the resin was filtered and washed with CH2Cl2. The filtrate was concentrated under reduced pressure. The obtained crude compounds are purified by flash chromatography (F.C.) or preparative reversed-phase HPLC.

XIII.7.8.1 3,4-DIHYDRO-4,5-DIMETHYLBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VII.34

F.C.: dichloromethane/MeOH 98/2 H O N CYC yield: 65% (white glass), 12 mg from 20 mg XIII.15

N Molecular Formula: C12H14N2O2 O Molecular weight: 218.25 g mol-1.

LC-MS: Peak at 3.8 min; ES-MS positive mode [m/z (fragment, intensity)]: 437.2 (2M+H+, 100), + + 260.2 (M+CH3CN+H , 30), 219.1 (M+H , 45).

+ + HRMS (ESI): Peak for C28H33N2O4 [M+H ] calculated: 219.1133, found: 219.1137.

1 H NMR (300 MHz, Acetone-d6): presence of two inversed boat-type conformers (ratio 60/40): major conformer: (P)-(R)-IX.24: δ 8.50 (br.s, NH), 7.41-7.30 (m, 3H), 7.22 (d, J= 8.9 Hz, 1H), 4.09 (ddq, J= 6.4/(3x)6.4/12.4 Hz, 1H), 2.96 (s, 1.8H), 2.83 (br.dd, J=16.3/5.8 Hz, 1H), 2.63 (dd, J=16.5/12.1Hz, 1H), 1.20 (d, J=6.7Hz, 3H). Minor conformer (M)-(R)-IX.24: 8.78 (br.s, NH), 7.50-7.43 (m, 3H), 7.16 (d, J= 8.3 Hz, 1H), 3.86 (ddq, J=11.2/8.5/(x3)6.6 Hz, 1H), 3.06 (s, 3H), 2.40 (dd, J=12.5/8.5 Hz, 1H), 2.25 (dd, J=12.5/11.3Hz, 1H), 1.15 (d, J=6.6 Hz, 3H) ppm.

270

Part 3: Experimental Procedures

O Me O N HN Me H H H H Me HN H H N O O Me (P)-(R)-IX.24 (M)-(R)-IX.24

13 C NMR (75 MHz, Acetone-d6): two inverted boat-type conformers are present on NMR-time scale: individual signals cannot be assigned ; all signals are listed: δ 138.9 (C), 137.0 (C), 135.4 (C), 131.6 (CH), 131.5 (CH), 131.3 (CH), 128.8 (CH), 128.6 (CH), 128.3 (CH), 127.7 (CH), 126.6

(CH), 56.0 (CH2), 51.7 (CH2), 44.6 (CH2), 41.8 (CH2), 37.8 (CH3), 26.5 (CH3), 21.1 (CH3), 19.9

(CH3) ppm.

IR (HATR): 3226 (w), 3085 (w), 2989 (w), 2943 (w), 1659 (m), 1651 (m), 1614 (s), 1591 (s), 1574 (m), 1494 (w), 1471 (m), 1443 (m), 1427 (m), 1402 (m), 1357 (s), 1343 (m), 1291 (w), 1251 (w), 1233 (m), 1186 (w), 1169 (w), 1119 (w), 1106 (w), 1093 (w), 1066 (w), 1038 (w), 1021 (w),1010 (w), 949 (w), 884 (w), 876 (w), 778 (w), 760 (s), 712 (m), 702 (m), 672 (w), 656 (w), 628 (w) cm- 1.

TLC: Rf = 0.14 (Dichloromethane/MeOH 98/2).

XIII.7.8.2 5-BENZYL-3,4-DIHYDRO-4-METHYLBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)- DIONE VII.35

F.C.: hexane/EtOAc/AcOH 50/50/1 H O N CYC yield: 45% (white glass), 28 mg from 66 mg XIII.16

N Molecular Formula: C18H18N2O2 O Molecular weight: 294.34 g mol-1.

LC-MS: Peak at 4.9 min; ES-MS positive mode [m/z (fragment, intensity)]: 0-100 % acetonitrile + + + in 6 min. 589.2 (2M+H , 65), 336.1 (M+CH3CN+H , 10), 295.1 (M+H , 100).

+ + HRMS (ESI): Peak for C18H19N2O2 [M+H ] calculated: 295.1446, found: 295.1452.

271

Part 3: Experimental Procedures

1 H NMR (400 MHz, CDCl3): presence of two inversed boat-type conformers: (ratio 60/40, individual assignment not possible): δ 8.00 and 7.84 (2x br.s, NH, ratio 6/4), 7.61 (dd, J=1.4/7.1 Hz, 0.6H), 7.57 (dd, J= 1.6/7.0 Hz, 0.4H), 7.50-7.21 (m, 7H), 7.17 (br.d, J= 7.1Hz, 0.4H), 7.10 (d, J= 7.5 Hz, 0.6H), 4.99 (d, J= 15.2 Hz, 0.4H), 4.83 (d, J= 14.5 Hz, 0.6H), 4.71 (d, J= 14.5 Hz, 0.6H), 4.58 (d, J= 15.6 Hz, 0.4H), 4.25-4.10 (m, 0.4H), 4.00-3.82 (m, 0.6H), 2.80-2.20 (m, 2H), 1.07 (d, J= 6.7 Hz, 1H), 0.99 (d, J= 6.6Hz, 2H) ppm.

13 C NMR (100 MHz, CDCl3): two inverted boat-type conformers are present on NMR-time scale: individual signals cannot be assigned ; all signals are listed: δ 172.1 (C), 169.8 (C), 168.8 (C), 138.4 (C), 137.0 (C), 136.8 (C), 134.9 (C), 134.0 (C), 132.7 (C), 130.8 (CH), 129.9 (CH), 128.9 (CH), 128.6 (CH), 128.3 (CH), 128.1 (CH), 127.9 (CH), 127.7 (CH), 127.2 (CH), 127.2 (CH),

127.0 (CH), 126.1 (CH), 125.4 (CH), 52.9 (CH2), 52.1 (CH), 52.3 (CH), 44.4 (CH2), 44.1 (CH2),

40.8 (CH2), 21.4 (CH3), 20.3 (CH3) ppm.

IR (HATR): 3395 (w), 3061 (w), 2971 (w), 2934 (w), 1668 (s), 1600 (s), 1540 (w), 1494 (m), 1477 (m), 1449 (m), 1417 (m), 1374 (m), 1347 (m), 1207 (m), 1295 (w), 1252 (w), 1237 (w), 1201 (m), 1167 (m), 1105 (m), 1079 (m), 1022 (s), 977 (w), 925 (w), 917 (w), 876 (vw), 848 (vw), 779 (m), 760 (m), 725 (m), 697 (m), 676 (w), 632 (w), 610 (w) cm-1.

TLC: Rf = 0.07 (hexane/EtOAc/AcOH 85/15/1)

XIII.7.8.3 3,4-DIHYDRO-8-METHOXY-4-METHYL-5-(3- (TRIFLUOROMETHYL)BENZYL)BENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VII.36

F.C.: Hexane/EtOAc/AcOH 50/50/1 H O N CYC yield: 23% (yellow glass), 4.5 mg from 20 mg XIII.17

MeO N Molecular Formula: C20H19F3N2O3 O Molecular weight: 392.37 g mol-1.

CF3 LC-MS: Peak at 5.6 min; ES-MS positive mode [m/z (fragment, intensity)]: 785.2 (2M+H+, 70), 393.1 (M+H+, 100).

+ + HRMS (ESI): Peak for C20H20F3N2O3 [M+H ] calculated: 393.1426, found: 393.1430.

272

Part 3: Experimental Procedures

1 H NMR (300 MHz, CDCl3): presence of two inversed boat-type conformers: (ratio 60/40, individual assignment not possible): δ 7.66-7.44 (m, 5H), 7.18-6.94 (m, 3H), 5.03 (d, J= 15.6 Hz, 0.40H), 4.99 (d, J= 14.9 Hz, 0.60H), 4.61 (d, J= 14.7 Hz, 0.60H), 4.58 (d, J= 16.0 Hz, 0.40H), 4.30-4.10 (m, 0.40H), 3.89 (s, 1.2H), 3.87 (s, 1.8H), 2.94-2.80 (m, 0.6H), 2.85-2.25 (m, 2H), 1.08 (d, J=6.5 Hz, 1.2 H), 1.08 (d, J=6.6 Hz, 1.8H) ppm.

13 C NMR (75 MHz, CDCl3): two inverted boat-type conformers are present: major conformer: δ 172.7 (CO), 171.1 (CO), 159.3 (C), 139.3 (C), 137.7 (C), 131.8 (CH), 129.2 (CH), 127.0 (CH),

125.3 (d, J=3.9 Hz, CH), 124.7 (d, J=3.8 Hz, CH), 117.5 (CH), 113.9 (CH), 55.8 (CH3), 52.8 (CH),

52.7 (CH2), 40.4 (CH2), 21.6 (CH3) ppm.

IR (HATR): 3191 (w), 3064 (w), 2926 (w), 1653 (m), 1637 (s), 1617 (m), 1540 (w), 1493 (m), 1473 (w), 1448 (m), 1436 (w), 1417 (w), 1405 (w), 1382 (w), 1340 (m), 1325 (s), 1287 (m), 1274 (m), 1223 (m), 1194 (m), 1157 (m), 1116 (s), 1075 (s), 1037 (m), 1022 (m), 940 (w), 923 (w), 890 (w), 811 (m), 799 (m), 770 (w), 749 (w), 744 (w), 701 (w). 659 (w), 643 (w), 620 (w) cm-1.

TLC: Rf = 0.40 (hexane/EtOAc/AcOH 50/50/1).

XIII.7.8.4 5-(CARBOXYBENZYLAMINOBUT-4-YL)-3,4-DIHYDRO-8-METHOXY-4- METHYLBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VII.37

F.C.: Dichloromethane/MeOH 99/1 to 98/2 H O N CYC yield: 17% (yellow oil), 5 mg from 30 mg XIII.18

MeO N Molecular Formula: C24H29N3O5 O Molecular weight: 439.51 g mol-1.

NH LC-MS: Peak at 5.3 min; ES-MS positive mode [m/z (fragment, O + O intensity)]: 440.2 (M+H , 100).

+ + HRMS (ESI): Peak for C24H30N3O5 [M+H ] calculated: 440.2185, found: 440.2191.

1 H NMR (400 MHz, CDCl3): presence of two inversed boat-type conformers: (ratio ~ 67/33, individual assignment not possible): δ 7.44 (br.s, 0.67NH), 7.40-7.28 (m, 5H), 7.18 (br.s, 0.33NH), 7.07 (d, J=8.4 Hz, 0.35H), 7.03-6.88 (m, 2.67H), 5.39 (br.s, 0.67NH), 5.25 (br.s, 0.33H), 5.19-

273

Part 3: Experimental Procedures

5.05 (m, 2H), 4.23-4.11 (m, 0.67H), 4.10-3.98 (m, 0.33H), 3.84 (s, 3H), 3.87-3.74 (m, 0.67H), 3.69-3.58 (m, 0.33H), 3.45-3.10 (m, 0.33H), 2.98-2.85 (m, 0.67H), 1.67-1.48 (m, 4H), 1.21 (d, J=

7.1 Hz, 1.00H), 1.17 (d, J = 6.7 Hz, 2.00H) ppm.

13 C NMR (100 MHz, CDCl3): two inverted boat-type conformers are present: individual signals cannot be assigned; all signals are listed: δ 168.1 (CO), 159.2 (C), 138.5 (C), 136.9 (C), 128.4 (CH), 128.0 (CH), 128.0 (CH), 128.0 (CH), 127.0 (CH), 126.6 (C), 125.0 (C), 117.6 (CH), 116.5

(CH), 113.5 (CH), 66.4 (CH2), 55.7 (CH3), 52.9 (CH), 48.4 (CH2), 40.7 (CH2), 27.4 (CH2), 26.8

(CH2), 25.1 (CH2), 21.5 (CH3) ppm.

IR (HATR): 3312 (w), 3245 (w), 3064 (w), 2920 (m), 2868 (w),

1715 (s), 1697 (s), 1669 (s), 1651 (s), 1622 (s), 1615 (s), 1555 (m), 1540 (m), 1522 (m), 1496 (s), 1473 (s), 1454 (s), 1436 (m), 1374 (m), 1338 (m), 1320 (m), 1289 (m), 1233 (s), 1173 (m), 1132 (m), 1088 (w), 1027 (m), 915 (vw), 871 (vw), 855 (w), 814 (w). 775 (w), 736 (w), 698 (w), 664 (w), 654 (w), 600 (w) cm-1.

TLC: Rf = 0.16 (Dichloromethane/MeOH 95/5).

XIII.7.8.5 4-BENZYL-3,4-DIHYDRO-5-METHYLBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)- DIONE VII.38

F.C.: dichloromethane/MeOH 99/1 H O N CYC yield: 46% (colorless glass), 5 mg from 10 mg XIII.19

N Molecular Formula: C H N O O 18 18 2 2 Molecular weight: 294.34 g mol-1.

LCMS: Peak at 5.2 min, ES-MS positive mode [m/z (fragment, intensity)]: 589.3 (2M+H+, 100), + + 336.1 (M+CH3CN+H , 10), 295.1 (M+H , 100).

+ + HR-MS (ESI): Peak for C18H19N2O2 [M+H ] calculated: 295.1446, found: 295.1437.

1 H NMR (400 MHz, CDCl3): presence of two inversed boat-type conformers: (ratio ~ 50/50, individual assignment not possible): δ 7.55-6.90 (m, 9H), 4.34-4.25 (m, 0.47H), 4.10-3.90 (m,

274

Part 3: Experimental Procedures

0.53H), 3.3 (s, 1.47H), 2.95 (s, 1.53H), 2.93-2.74 (m, 1.59H), 2.67 (dd, J= 6.8/13.4 Hz, 1.41H) 2.43 (d, J= 9.9 Hz, 1H) ppm.

13 C NMR (100 MHz, CDCl3): two inverted boat-type conformers are present: individual signals cannot be assigned; all signals are listed: δ 172.0 (CO), 170.3 (CO), 169.1 (CO), 168.4 (CO), 137.0 (C), 136.3 (C), 136.3 (C), 135.0 (C), 133.7 (C), 132.7 (C), 130.9 (CH), 130.8 (CH), 130.1 (CH), 129.1 (CH), 128.9 (CH), 128.8 (CH), 128.6 (CH), 128.2 (CH), 128.2 (CH), 127.6 (CH), 127.2

(CH), 127.1 (CH), 126.3 (CH), 125.3 (CH), 61.0 (CH), 55.4 (CH), 41.7 (CH2), 41.3 (CH2), 39.4

(CH2), 39.0 (CH3), 38.7 (CH2), 26.6 (CH3) ppm.

IR (HATR): 3201 (w), 3074 (w), 2940 (w), 2873 (w), 1664 (m), 1635 (m), 1620 (m), 1602 (s), 1496 (w), 1480 (m), 1457 (w), 1431 (w), 1401 (m),1374 (w), 1207 (m), 1153 (s), 1108 (m), 1072 (m), 1057 (m), 1028 (m), 1002 (m), 811 (w), 778 (w), 754 (m), 700 (m), 667 (w), 625 (w), 615 (vw) cm-1.

TLC: Rf = 0.12 (Dichloromethane/MeOH 97/3).

XIII.7.8.6 4,5-DIBENZYL-3,4-DIHYDROBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VII.39

F.C.: hexane/EtOAc/AcOH 50/50/1 H O N CYC yield: 48% (colorless glass), 14 mg from 35 mg XIII.20

N Molecular Formula: C24H22N2O2 O Molecular weight: 370.44 g mol-1.

LC-MS: Peak at 5.8 min; ES-MS positive mode [m/z (fragment, intensity)]: 741.3 (2M+H+, 20), 371.2 (M+H+, 100).

+ + HRMS (ESI): Peak for C24H23N2O2 [M+H ] calculated: 371.1759, found:.371.1750

1 H NMR (400 MHz, CDCl3): presence of two inversed boat-type conformers: (ratio 80/20, individual assignment not possible): δ 7.61-7.31 (m, 8H), 7.26-7.09 (m, 4H), 6.86 (d, J=7.3Hz,

2H), 4.92 (d, J= 14.4Hz, ~0.8H), 4.80(s, ~0.4H), 4.49-4.34 (m, ~0.2H), 4.28 (d, J= 14.4 Hz, ~0.8H), 4.08-3.95 (m, ~0.8H), 2.74-2.18 (m, 4H) ppm.

275

Part 3: Experimental Procedures

13 C NMR (100 MHz, CDCl3): two inverted boat-type conformers are present: major conformer: δ 171.9 (CO), 137.0 (C), 136.5 (C), 136.4 (C), 133.8 (C), 131.0 (CH), 130.1 (CH), 129.0 (CH), 129.0 (CH), 128.8 (CH), 128.7 (CH), 128.2 (CH), 127.9 (CH), 127.1 (CH), 125.4 (CH), 57.4 (CH), 53.4

(CH2), 42.5 (CH2), 38.8 (CH2) ppm

IR (HATR): 3317 (vw), 3060 (w), 3027 (w), 3002 (w), 2922 (w), 2852 (w), 1667 (s), 1626 (s), 1600 (m), 1491 (w), 1473 (m), 1450 (m), 1415 (w), 1383 (m), 1361 (w), 1340 (w), 1315 (m), 1291 (w), 1256 (w), 1238 (w), 1199 (w), 1161 (w), 1149 (w), 1105 (w), 1077 (w), 1043 (w), 1027 (w), 1003 (w), 974 (w), 951 (w), 935 (vw), 811 (w), 869 (vw), 845 (vw), 779 (m), 756 (m), 735 (s), 703 (m), 696 (m), 645 (w), 634 (w), 616 (w) cm-1.

TLC: Rf = 0.33 (hexane/EtOAc/AcOH 50/50/1).

XIII.7.8.7 4-BENZYL-8-BROMO-3,4-DIHYDRO-5-PENTYLBENZO[g][1,5]DIAZOCINE- 2(1H),6(5H)-DIONE VII.40

F.C.: Dichloromethane/MeOH 99/1 to 98/2 H O N CYC yield: 31% (white powder), 9 mg from 30 mg XIII.21

Br N Molecular Formula: C22H25BrN2O2 O Molecular weight: 429.35 g mol-1.

LC-MS: Peak at 6.4 min; ES-MS positive mode [m/z (fragment, intensity)]: 859.0 (2M(79Br)+H+, 50), 432.1(M(81Br)+H+, 30), 431.1 (M(81Br), 30), 430.0 (M(79Br)+H+, 24), 429.1 (M(79Br), 100)

79 + + HRMS (ESI): Peak for C22H26 BrN2O2 [M+H ] calculated: 429.1177, found: 429.1172.

1 H NMR (400 MHz, CDCl3): presence of two inversed boat-type conformers: (ratio 84/16, individual assignment not possible): δ 7.59-7.20 (m, 6H), 7.08-6.86 (m, 3H), 4.33-4.22 (m, 0.16H), 4.10-3.90 (m, 1.68H), 3.67-3.54 (m, 0.16H), 3.33-3.21 (m, 0.16H), 2.99 (dd, J=14.0/7.8 Hz, 0.16H), 2.86-265 (m, 2H), 2.63-2.52 (m, 0.84H), 2.51-2.35 (m, 1.68H), 1.60-1.15 (m, 6H), 0.93 (t, J=7.0 Hz, 0.48H), 0.88 (t, J = 7.1 Hz, 2.52H) ppm.

276

Part 3: Experimental Procedures

13 C NMR (100 MHz, CDCl3): two inverted boat-type conformers are present: major conformer: δ 171.8 (CO), 166.4 (C), 138.8 (C), 135.7 (C), 133.5 (CH), 132.7 (CH), 132.6 (C), 129.4 (CH), 128.9

(CH), 127.4 (CH), 126.5 (CH), 121.4 (C), 58.6 (CH), 50.3 (CH2), 41.8 (CH2), 38.5 (CH2), 29.0

(CH2), 27.0 (CH2), 22.3 (CH2), 14.0 (CH3) ppm.

IR (HATR): 3200 (w), 3064 (w), 3028 (w), 2956 (w), 2928 (w), 1676 (s), 1616 (s), 1474 (s), 1452 (m), 1363 (m), 1312 (m), 1232 (w), 1178 (w), 1146 (w), 1121 (w), 1101 (w), 1079 (w), 1049 (vw), 1028 (vw), 933 (vw), 915 (vw), 882 (w), 818 (w), 754 (w), 726 (w), 702 (m), 646 (vw), 618 (vw) cm-1.

TLC: Rf = 0.50 (Dichloromethane/MeOH 95/5).

XIII.7.8.8 4-BENZYL-3,4-DIHYDRO-5-(2-METHOXYETHYL)-9- (TRIFLUOROMETHYL)BENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VII.41

F.C.: Dichloromethane/MeOH 98/2 H O N F3C CYC yield: 56.7% (white glass), 18 mg from 30 mg XIII.22

N Molecular Formula: C21H21F3N2O3 O Molecular weight: 406.39 g mol-1. OMe LCMS: Peak at 5.7 min; ES-MS positive mode [m/z (fragment, intensity)]: 407.2 (M+H+, 100).

+ + HR-MS (ESI): Peak for C21H22F3N2O3 [M+H ] calculated: 407.1582, found: 407.1581.

1 H NMR (400 MHz, CDCl3): presence of two inversed boat-type conformers: (ratio 80/20, individual assignment not possible): δ 7.99 and 7.81 (2x br.s, NH, ratio 8/2), 7.68-7.50 (m, 2H), 7.42-7.18 (m, 4H), 7.07-6.92 (m, 2H), 4.34-4.00 (m, 2H), 3.65-3.45 (m, 2H), 3.38 (s, 0.66H), 3.33 (s, 2.34H), 3.10-2.30 (m, 5H) ppm.

13 C NMR (100 MHz, CDCl3): no multiple conformations observed: δ 171.8 (CO), 167.2 (C), 140.2 (C), 136.6 (C), 134.4 (C), 132.7 (q, J= 33.0 Hz, C) 130.6 (CH), 129.2 (CH), 128.9 (CH), 127.3

(CH), 124.7 (q, J= 3.7 Hz, CH), 123.0 (q, J= 272.9 Hz, C), 122.3 (q, J= 3.7 Hz, CH), 70.6 (CH2),

59.3 (CH3), 55.9 (CH), 50.2 (CH2), 42.1 (CH2), 38.8 (CH2) ppm.

277

Part 3: Experimental Procedures

IR (HATR): 3198 (w), 3121 (w), 3059 (w), 2981 (w), 2926 (w), 1688 (m), 1651 (w), 1605 (s), 1578 (w), 1513 (w), 1470 (w), 1453 (w), 1390 (w), 1371 (w), 1330 (s), 1299 (m), 1212 (w), 1173 (m), 1117 (s), 1080 (m), 1069 (m), 1016 (w), 997 (w), 959 (w), 921 (w), 892 (w), 876 (w), 838 (w), 813 (w), 778 (w), 755 (w), 735 (w), 717 (w), 701 (m), 670 (w), 642 (w) cm-1.

TLC: Rf = 0.22 (Dichloromethane/MeOH 98/2).

XIII.7.8.9 4-BENZYL-8-BROMO-3,4-DIHYDRO-5-(4- FLUOROBENZYL)BENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VII.42

F.C.: Dichloromethane/MeOH 98/2 H O N CYC yield: 23.9% (white powder), 7 mg from 30 mg XIII.23

Br N Molecular Formula: C24H20BrFN2O2 O Molecular weight: 467.33 g mol-1.

F LC-MS: Peak at 6.3 min; ES-MS positive mode [m/z (fragment, intensity)]: 937.0 (2M(81Br)+H+, 12), 935.0 (2M(79Br)+H+, 23), 470.0 (M(81Br)+H+, 28), 469.1 (M(81Br), 100), 468.1 (M (79Br)+H+, 28), 467.0 (M (79Br), 100).

79 + + HRMS (ESI): Peak for C24H21 BrFN2O2 [M+H ] calculated: 467.0770, found: 467.0774.

1 H NMR (400 MHz, CDCl3): presence of two inversed boat-type conformers: (ratio 87/13, individual assignment not possible): δ 7.70 (d, J=2.3 Hz, 0.13H), 7.62 (br.s, NH), 7.58 (dd, J=2.3/8.3 Hz, 0.13H), 7.53 (dd, J=2.3/8.4 Hz, 0.87H), 7.70 (d, J=2.2 Hz, 0.87H), 7.40-7.16 (m, 5H), 7.06-6.83 (m, 5H), 5.02 (d, J =14.5 Hz, 0.87H), 4.79 (d, J=15.5 Hz, 0.13H), 4.68 (d, J=15.6 Hz, 0.13H), 4.49-4.28 (m, 0.13H), 4.08 (d, J = 14.6 Hz, 1H,), 4.09-3.98 (m, 0.87H), 2.84 (dd, J=6.8 Hz, 0.13H), 2.75-2.61 (m, 2.13H), 4.45 (dd, J=12.7/11.6 Hz, 0.87H), 2.30 (br.dd, J=12.6/8.3 Hz, 0.87H) ppm.

13 C NMR (100 MHz, CDCl3): two inverted boat-like conformers are present: major conformer: δ 171.6 (CO), 167.3 (CO), 162.4 (d, J= 246.5 Hz, C), 138.0 (C), 135.6 (C), 133.9 (CH), 133.0 (CH), 132.7 (C), 132.0 (d, J= 2.9 Hz, C), 130.5 (d, J= 8.1 Hz, CH), 129.3 (CH), 128.9 (CH), 127.5 (CH),

278

Part 3: Experimental Procedures

126.5 (CH), 121.5 (C), 115.6 (d, J=21.3 Hz, CH), 57.7 (CH), 52.7 (CH2), 41.8 (CH2), 38.3 (CH2) ppm.

IR (HATR): 3198 (w), 3062 (w), 3028 (w), 2922 (w), 1676 (s), 1615 (s), 1605 (s), 1508 (s), 1471 (s), 1453 (m), 1415 (m), 1364 (m), 1343 (m), 1307 (m), 1261 (w), 1219 (s), 1179 (w), 1155 (m), 1109 (w), 1095 (w), 1080 (w), 1049 (w), 1028 (w), 1015 (w), 970 (w), 933 (vw), 884 (w), 849 (w), 821 (m), 777 (w), 754 (m), 731 (w), 702 (s), 656 (w), 647 (w), 614 (vw) cm-1.

TLC: Rf = 0.19 (Dichloromethane/MeOH 98/2).

XIII.7.8.10 3,4-DIHYDRO-4-ISOBUTYL-5-METHYLBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)- DIONE VII.43

F.C.: Dichloromethane/MeOH 99/1 to 98/2 H O N CYC yield: 45% (colorless glass), 13 mg from 30 mg XIII.24

N Molecular Formula: C15H20N2O2 O Molecular weight: 260.33 g mol-1.

LC-MS: Peak at 4.8 min, ES-MS positive mode [m/z (fragment, intensity)]: 521.2 (2M+H+, 100), + + 302 (M+ CH3CN+H , 10), 261.1 (M+H , 40).

+ + HRMS (ESI): Peak for C15H21N2O2 [M+H ] calculated: 261.1603, found: 261.1610.

1 H NMR (400 MHz, CDCl3): presence of two inversed boat-type conformers: (ratio 75/25, individual assignment not possible): δ 7.64 (br.s, NH), 7.54-7.37 (m, 3H), 7.17 (br.dd, J= 1/7.8 Hz, 0.75H), 7.10 (br.dd, J= 1.2/7.9 Hz, 0.25H), 3.95 (dq, 4.5/9.4 Hz, 0.75H), 3.85-3.70 (m, 0.25H), 3.18 (s, 0.77H), 3.00 (s, 2.23H), 2.71 (d, J=9.0 Hz, 1.46H), 2.52 (dd, J=12.4/9.0 Hz, 0.27H), 2.22 (dd, J=12.3/10.7 Hz, 0.25H), 1.65-0.96 (m, 3H), 0.88 (d, J=6.4 Hz, 0.8H), 0.88 (d, J=6.4 Hz, 0.88H), 0.85 (d, J=6.7 Hz, 2.2 Hz), 0.65 (d, J= 6.5 Hz, 2.2H) ppm.

13 C NMR (100 MHz, CDCl3): two inverted boat-like conformers are present: major conformer: δ 170.5 (C), 169.4 (C), 135.0 (C), 132.8 (C), 130.8 (CH), 128.3 (CH), 127.5 (CH), 126.2 (CH), 52.6

(CH), 45.3 (CH2), 41.9 (CH2), 26.2 (CH), 24.5 (CH3), 23.0 (CH3), 21.3 (CH3) ppm.

279

Part 3: Experimental Procedures

IR (HATR): 3483 (w) 3063 (w), 2954 (w), 2923 (w), 2869 (w), 1623 (br.s), 1602 (s), 1540 (w), 1468 (m), 1454 (m), 1426 (m), 1401 (m), 1365 (s), 1287 (w), 1265 (w), 1234 (m), 1177 (w), 1116 (w), 1070 (w), 1031 (w), 990 (vw), 953 (w), 926 (vw), 892 (vw), 847 (vw), 779 (w), 758 (m), 711 (w), 660 (w), 628 (w), 614 (w) cm-1.

TLC: Rf = 0.27 (Dichloromethane/MeOH 95/5).

XIII.7.8.11 5-BENZYL-3,4-DIHYDRO-4-ISOBUTYLBENZO[g][1,5]DIAZOCINE-2(1H),6(5H)- DIONE VII.44

F.C.: Dichloromethane/MeOH 99/1 to 98/2 H O N CYC yield: 45% (colorless glass), 13 mg from 30 mg XIII.25

N Molecular Formula: C21H24N2O2 O Molecular weight: 336.42 g mol-1.

LC-MS: Peak at 5.8 min; ES-MS positive mode [m/z (fragment, intensity)]: 673.3 (2M+H+, 80), 337.1 (M+H+, 100).

+ + HRMS (ESI): Peak for C21H25N2O2 [M+H ] calculated: 337.1916, found: 337.1926.

1 H NMR (400 MHz, CDCl3): presence of two inversed boat-type conformers: (ratio ~ 65/35, individual assignment not possible): δ 7.69 (br.s, NH), 7.62-7.21 (m, 8H), 7.16 (dd, J= 1.7/7.8 Hz, 0.4H), 7.09 (dd, J=1.5/7.8 Hz, 0.6H), 4.89 (d, J=14.2 Hz, 0.65H), 4.80 (d, J=15.1 Hz, 0.35H), 4.66 (d, J=15.1 Hz, 0.35H), 4.61 (d, J=14.4 Hz, 0.65H), 4.15-4.01 (m, 0.35H), 3.85-3.72 (m, 0.65H), 2.66-2.10 (m, 2H), 1.64-0.96 (m, 3H), 0.72 (d, J=6.4 Hz, 2H), 0.69 (d, J=6.3 Hz, 2H), 0.58 (d, J=6.3 Hz, 1H), 0.56 (d, J=6.4 Hz, 0.1H) ppm.

13 C NMR (100 MHz, CDCl3): two inverted boat-like conformers are present: major conformer: δ 172.3 (CO), 170.3 (CO), 168.9 (C), 137.2 (C), 136.6 (C), 133.9 (C), 130.8 (CH), 129.0 (CH), 128.6

(CH), 128.2 (CH), 128.1 (CH), 127.8 (CH), 125.4 (CH), 54.0 (CH), 53.2 (CH2), 45.8 (CH2), 39.1

(CH2), 24.5 (CH), 23.4 (CH3), 20.9 (CH3) ppm; minor conformer: δ 170.4 (CO), 138.3 (C), 135.0

(C), 132.7 (C), 127.5 (CH), 127.5 (CH), 126.1 (CH), 53.8 (CH), 44.5 (CH2), 42.7 (CH2), 42.1

(CH2), 24.3 (CH), 22.3 (CH3), 21.4 (CH3) ppm.

280

Part 3: Experimental Procedures

IR (HATR): 3197 (w), 3061 (w), 2954 (w), 2919 (w), 2868 (w), 1672 (s), 1620 (s), 1600 (s), 1494 (m), 1477 (m), 1470 (m), 1452 (m), 1416 (m), 1370 (s), 1351 (m), 1323 (m), 1301 (m), 1287 (w), 1248 (m), 1203 (w), 1184 (w), 1161 (m), 1104 (w), 1077 (w), 1028 (w), 1001 (w), 977 (w), 950 (w), 918 (w), 882 (vw), 847 (w), 816 (w), 779 (m), 760 (s), 723 (m), 697 (s), 632 (w), 627 (w), 614 (vw) cm-1.

TLC: Rf = 0.34 (Dichloromethane/MeOH 95/5).

XIII.8 SYNTHESIS OF N-FMOC-3-AMINO-2,2-DIALKYLPROPIONIC ACID

XIII.8.1 Synthesis of METHYL 2-CYANO-2,5-(DIMETHYL)METHYL PENTANOATE VIII.11

1 eq K2CO3, 1 eq MeI, 0.1 M DMF, 1 hr Me NC COOMe NC COOMe

VIII.10 VIII.11

To a cooled (0 °C) solution of nitrile VIII.10 (0.99 mmol, 1 eq) in THF (10 ml) is added K2CO3 (0.99 mmol, 1 eq). After 30 min, MeI (0.99 mmol, 1 eq) is added dropwise, and the reaction mixture is stirred for 1 hour. Ammonium chloride is added and the reaction mixture is extracted using ethylacetate (3x). The combined organic layers are concentrated under reduced pressure after which the residue is purified by flash chromatography in (hexane/ethylacetate 95/5).

F.C.: hexane/EtOAc 95/5.

Me Yield: 50% (colorless oil) NC COOMe Molecular Formula: C9H15NO2

Molecular Weight: 169.22 g mol-1

LC-MS: no ionization

GC-MS: Peak at 9.9 min, ES-MS negative mode [m/z (fragment, intensity)]: 168 (M-H+), 154 (M- Me-H+), 127 (M-Me -CN-H+).

281

Part 3: Experimental Procedures

1 H NMR: (300 MHz, CDCl3): δ 3.82 (s, 3H), 1.94-1.66 (m, 3H), 1.61 (s, 3H), 1.02 (d, J= 6.6 Hz, 3H), 0.91 (d, J= 6.4 Hz, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 170.4 (CO), 120.1 (CN), 53.4 (CH3), 46.5 (CH2), 42.9 (C), 26.1

(CH), 25.1 (CH3), 23.3 (CH3), 22.3 (CH3) ppm.

IR (HATR): 2959 (w), 2876 (vw), 2243 (vw), 1743 (s), 1626 (w), 1457 (m), 1436 (w), 1390 (w), 1306 (w), 1284 (w), 1237 (s), 1185 (m), 1150 (m), 1084 (w), 986 (w), 980 (w), 929 (w), 850 (w), 820 (w), 802 (w), 765 (w), 647 (vw), cm-1.

TLC: Rf =0.19 (hexane/ EtOAc 95/5)

XIII.8.1 Synthesis of METHYL 2-CYANO-2-(NAPHTH-2-YL)METHYL)PROPIONATE VIII.13

5 eq MeI, 2eq NaOMe, THF 1M, 1h. Me NC COOMe NC COOMe

VIII.12 VIII.13

To a cooled (0 °C) solution of nitrile VIII.12 (4.18 mmol, 1eq) in THF (100 ml) is added NaOMe (8.38 mmol, 2 eq). After 30 min, MeI (20.9 mmol, 5 eq) is added dropwise, and the reaction mixture is stirred for 35 min. Water is added and the reaction mixture is extracted using ethylacetate (3x). The combined organic layers are concentrated under reduced pressure after which the residue is purified by flash chromatography in (hexane/ethylacetate 95/5).

F.C.: hexane/EtOAc 95/5.

Yield: 78% (yellow oil) Me NC COOMe Molecular Formula: C16H15NO2

Molecular Weight: 253.29 g mol-1

LC-MS: Peak at 6.4 min, ES-MS negative mode [m/z (fragment, intensity)]: 252.2 (M-H+, 5), 329.2 (M, 20).

282

Part 3: Experimental Procedures

+ + HRMS (ESI): C16H19N2O2 [M+NH4 ]: calculated 271.1446; Found: 271.1444.

1 H NMR: (300 MHz, CDCl3): δ 7.86-7.82 (m, 3H), 7.75 (br.s, 1H), 7.53-7.47 (m, 2H), 7.41 (dd, 1H, J=8.5/1.9 Hz), 3.75 (s, 3H), 3.42 (d, J = 13.4 Hz, 1H), 3.23 (d, J= 13.4 Hz, 1H), 1.69 (s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 169.5 (CO), 133.2 (C), 132.8 (C), 131.6 (C), 129.0 (CH), 128.3

(CH), 127.8 (CH), 127.6 (CH), 126.3 (CH), 126.1 (CH), 119.6 (CN), 53.4 (CH3), 45.3 (C), 43.8

(CH2), 23.2 (CH3) ppm.

IR (HATR): 3054 (w), 2987 (w), 2964 (w), 2953 (w), 1741 (s, sharp), 1633 (w), 1699 (w), 1508 (w), 1453 (m), 1434 (m), 1379 (w), 1322 (w), 1283 (m), 1253 (s), 1232 (s), 1202 (m), 1156 (m), 1115 (m), 1090 (w), 1017 (w), 984 (w), 897 (w), 859 (m), 822 (m), 783 (m), 748 (s), 663 (w), 634 (vw), 623 (w) cm-1.

TLC: Rf =0.16 (hexane/ EtOAc 95/5)

XIII.8.2 Synthesis of METHYL 2-METHYL-3-(NAPHTH-2-YLMETHYL)-3-(TERT- BUTOXYCARBONYL)AMINOPROPIONATE VIII.14

0.1 eq NiCl2.6H2O, 7 eq NaBH4, 2 eq Boc2O, Boc 0.2 M MeOH, 0 to r.t HN NC COOMe COOMe

VIII.13 VIII.14

To a cooled (0 °C) solution of the nitrile (10.06 mmol, 1 eq) in MeOH (82 ml) is added Boc2O

(20.12 mmol, 2 eq) and NiCl2.6H2O (0.12 mmol, 0.1 eq). Afterwards NaBH4 (70.42 mmol, 7 eq) is added protionwise (0.22 eq every 10 min). After the addition, the reaction mixture is stirred overnight under N2 atmosphere, after which it is concentrated under reduced pressure. The residue is dissolved in ethyl acetate. After washing with a saturated aqueous solution of NaHCO3, the organic layer is dried over magnesium sulfate. The drying agent is filtered, the solvent is removed under reduced pressure and the residue is purified by flash chromatography.

283

Part 3: Experimental Procedures

F.C.: hexane/EtOAc 95/5 to 9/1.

O O Yield: 60% (yellow oil) HN COOMe Molecular Formula: C21H27NO4

Molecular Weight: 357.44 g mol-1

LC-MS: Peak at 6.1 min; ES-MS positive mode [m/z (fragment, intensity)]: 258.1 (M M- + - isobutene-CO2+H , 100), 226.1 (M-isobutene-CO2-OMe , 18), 197.1 (18).

+ + HRMS (ESI): C16H20NO2 [M-isobutene-CO2+H ]: calculated 258.1494; Found: 258.1501.

1 H NMR: (300 MHz, CDCl3): δ 7.84-7.72 (m, 3H), 7.57 (br.s, 1H), 7.50-7.41 (m, 2H), 7.22 (dd, J=8.4/1.7 Hz, 1H), 4.97 (br.s, NH), 3.69 (s, 3H), 3.45-3.30 (m, 2H), 3.08 (d, J= 13.6 Hz, 1H), 3.00 (d, J= 13.4 Hz, 1H), 1.46 (s, 9H), 1.24 (s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 176.4 (CO), 156.1 (CO), 134.1 (C), 133.2 (C), 132.3 (C), 128.6 (CH), 128.2 (CH), 127.7 (CH), 127.6 (CH), 127.6 (CH), 126.0 (CH), 125.5 (CH), 79.2 (C), 51.9

(CH3), 48.7 (CH2), 47.0 (C), 43.3 (CH2), 28.3 (3XCH3) 20.3 (CH3) ppm.

IR (HATR): 3372 (w), 3052 (w), 2975 (w), 1713 (s, sharp), 1633 (vw), 1600 (w), 1505 (m), 1454 (m), 1391 (w), 1364 (m), 1332 (w), 1253 (m), 1245 (m), 1165 (s), 1042 (w), 1016 (w), 989 (vw), 952 (w), 915 (w), 897 (w), 856 (m), 823 (m), 802 (m), 779 (w), 654 (w), 634 (w), 621 (w), 609 (w) cm-1.

TLC: Rf =0.10 (hexane/ EtOAc 9/1).

284

Part 3: Experimental Procedures

XIII.8.3 Synthesis of 2-METHYL-2-(NAPHTH-2-YLMETHYL)-2-(TERT- BUTOXYCARBONYL)AMINOPROPIONIC ACID VIII.16

Boc 4 eq NaOH, H2O/MeOH (2/1), 50°C, 2 h Boc HN HN COOMe COOH

VIII.14 VIII.16

To a solution of methyl ester (8.3 mmol, 1 eq) in H2O/MeOH (2/1) (83 ml), is added NaOH (16.6 mmol, 2 eq). The reaction mixture is refluxed at 50° C for 2 h, after which it is acidified with a 1

M aqueous KHSO4 solution to pH 2. The product is extracted with ethyl acetate (600 ml) and filtered over silica, the residue is purified by column chromatography

F.C (hexane/EtOAc/AcOH 97/3/1)

Yield: 97% (yellow oil) H O N COOH Molecular Formula: C20H25NO4 O Molecular Weight: 343.41 g mol-1

LC-MS: Peak at 4.9 min; ES-MS negative mode [m/z (fragment, intensity)]: 342.1 (M-H+, 100), + 268.1 (M-isobutene-H2O-H , 25).

- + HRMS (ESI): C20H24NO4 [M-H ]: calculated 342.1705; Found: 342.1710.

1 H NMR: (300 MHz, CDCl3): presence of two carbamate rotamers on NMR-time scale: (ratio ~ 40/60): δ 7.82-7.73 (m, 3H), 7.63 (br.s, 1H), 7.49-7.42 (m, 2H), 7.30 (dd, 1H, J = 1.6/8.5 Hz), 6.28 and 5.03 (2x br.s, NH, ratio 40/60), 3.78-3.55 (m, 0.37H), 3.41-3.35 (m, 0.63H), 3.10 (br.s, 2H), 1.45 (s, 9H), 1.26 (s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): presence of two carbamate rotamers on NMR-time scale: δ 177.7 (CO), 177.8 (CO), 157.8 (CO), 155.9 (CO), 135.7 (C), 135.6 (C), 133.5 (C), 132.3 (C), 128.3 (CH), 127.6 (CH), 127.5 (CH), 127.5 (CH), 127.4 (CH), 127.3 (CH), 127.3 (CH) 127.1 (CH), 127.0

(CH), 126.1 (CH), 125.5 (CH), 81.2 (C), 79.7 (C), 47.4 (CH3), 47.1 (CH3), 42.2 (CH2), 41.4 (C),

35.9 (CH2), 28.3 (CH3), 28.2 (CH3), 28.2 (CH3) ppm.

285

Part 3: Experimental Procedures

IR (HATR): 3457 (vw), 3323 (vw), 3052 (w), 2975 (w), 2929 (w), 2915 (w), 1697 (s, sharp), 1600 (w), 1507 (m), 1454 (m), 1421 (m), 1392 (m), 1365 (m), 1332 (w), 1270 (m), 1248 (m), 1163 (s), 1041 (w), 1016 (m), 950 (w), 910 (w), 895 (w), 856 (m), 821 (m), 776 (m), 746 (m), 734 (w), 650 (w), 621 (w) cm-1.

TLC: Rf = 0.34 (hexane/ EtOAc/AcOH 7/3/1).

XIII.8.4 Synthesis of HCl salt of 2-METHYL-2-(NAPHTH-2- YLMETHYL)AMINOHYDROCHLORIDEPROPIONIC ACID VIII.17

Boc Dioxane/conc. HCl 9/1, r.t , 7 h. HN HCl.H2N COOH COOH

VIII.16 VIII.17

To a solution of Boc-amino acid (3.211 mmol, 1 eq), in dioxane (32 ml) is added concentrated HCl 4 ml. The obtained colorless solution is stirred for 7 h at room temperature, after which it is concentrated under reduced pressure to give the product as a white solid. The product is used as such in the next step.

XIII.8.5 Synthesis of 3-(9-FLUORENYLMETHYLOXYCARBONYL)AMINO-2-METHYL-2- (NAPHTH-2-YLMETHYL)PROPIONIC ACID VIII.8

H Me i) 2 eq TMSiCl, 1 eq DIPEA, CH Cl , reflux 6 h. HCl.H2N 2 2 N COOH ii) 2 eq DIPEA, 1 eq FmocCl, 0 C-r.t, o.n Fmoc COOH 44% VIII.17 VIII.8

To a mixture of crude amino acid hydrochloride (3.211 mmol, 1 eq) in dichloromethane (32 ml), is added DIPEA (3.211 mmol, 1 eq) and TMSCl (6.432 mmol, 2 eq). The mixture is refluxed for 6 hours then it is allowed to cool down to 0°C followed by addition of DIPEA (6.433 mmol, 2 eq) and FmocCl (3.211 mmol, 1eq). The reaction mixture is stirred overnight at room temperature.

286

Part 3: Experimental Procedures

The resulting white suspension is acidified using 6 M HCl to pH 1 and the resulting clear solution is extracted with EtOAc (3 x 50 ml). The combined organic phases are concentrated under reduced pressure. The obtained residue is further purified by flash chromatography.

F.C.: Petroleum ether/Acetone 99/1 to 8/2.

Yield: 44% (white powder) H O N COOH Molecular Formula: C30H27NO4 O Molecular Weight: 465.54 g mol-1

LC-MS: Peak at 5.7 min; ES-MS positive mode [m/z (fragment, intensity)]: 931.2 (2M+H+, 40), + 243.1 ([M-dibenzofluvene-CO2+H ], 100).

+ + HRMS (ESI): C30H28NO4 [M+H ]: calculated 466.2018, found: 466.2019.

1 H NMR: (300 MHz, Acetone-d6): δ 7.87-7.79 (m, 5H), 7.76 (s, 1H), 7.71 (d, J=7.5 Hz, 2H), 7.50- 7.38 (m, 5H), 7.34-7.29 (td, J =7.3/0.9 Hz, 2H), 6.4 (br.s., NH), 4.35 (d, J=6.9 Hz, 2H), 4.23 (t, J= 6.9 Hz, 1H), 3.49 (br.s, 2H), 3.23 (d, J= 13.3 Hz, 1H), 3.01 (d, J= 13.2 Hz, 1H), 1.20 (2x br.s, 3H) ppm.

13 C NMR: (75 MHz, Acetone-d6): δ 177.4 (CO), 158.1 (C), 145.6 (C), 142.6 (C), 136.5 (C), 134.9 (C), 133.8 (C), 130.1 (CH), 130.1 (CH), 129.0 (CH), 129.0 (CH), 128.9 (CH), 128.7 (CH), 128.4

(CH), 127.2 (CH), 126.8 (CH), 126.6 (CH), 121.3 (CH), 67.5 (CH2), 49.7 (CH2), 49.0 (C), 48.6

(CH), 43.8 (CH2), 20.9 (CH3) ppm.

IR(HATR): 3421 (w), 3320 (w), 3052 (w), 3023 (w), 2923 (m), 2853 (w), 2581 (w), 1953 (vw), 1697 (s, sharp), 1611 (w), 1600 (w), 1518 (m), 1507 (m), 1476 (w), 1462 (m), 1449 (m), 1415 (w), 1385 (w), 1359 (w), 1324 (w), 1224 (br.m), 1153 (m), 1140 (m), 1023 (w), 987 (w), 962 (w), 950 (w), 916 (w), 895 (w), 857 (w), 822 (m), 758 (m), 735 (s), 702 (w), 666 (w), 620 (w) cm-1.

TLC: Rf = 0.11 (Petroleum ether/Acetone 8/2).

287

Part 3: Experimental Procedures

XIII.9 SYNTHESIS OF 3,3-DISUBSTITUTED-1,5-BENZODIAZOCINE-2,6-DIONES

XIII.9.1 β2,2-Amino acid coupling on Wang resin

1. 2 eq Fmoc-AA-OH VIII.8, 2 eq DIC, O 0.2 eq DMAP, CH2Cl2, r.t, 24 h OH O NH2 2. Ac2O/DIPEA/CH2Cl2 (1/1/3), r.t, 2x1 h 3. 20% 4-methylpiperidine in DMF, 2x20 min OAc

VIII.6 VIII.19

2,2 Preactivation: To a cooled (0°C) solution of N-Fmoc-β -amino acid VIII.8 (2 eq) in CH2Cl2 (3 ml), DIC (0.970 mmol, 2 eq) is added. The reaction mixture is stirred for 20 minutes at 0 °C.

Coupling: The crude preactivation mixture is transferred to a solid-phase reaction vessel containing Wang resin (0.5 mmol, 1 eq, calculated from manufacturer’s loading 1.9 mmol/g), washed and preswollen with CH2Cl2) after which DMAP is added (0.0970 mmol, 0.2 eq). The suspension is shaken for 24 hours at room temperature after which the resin is filtered and washed consecutively with CH2Cl2 (3x), DMF (3x), MeOH (3x), and CH2Cl2 (3x).

Loading: The loading of the resulting resins was determined by Fmoc UV quantification: 0.4836 mmol/g. Yield of final product was calculated in reference to this loading values. Amount of resin for the following synthesis we used 0.0484 mmol of the prepared Fmoc-β2,2-amino acid-loaded Wang resin.

Deprotection: The resin is washed with DMF (3x) and treated with a solution of 20% 4- methylpiperidine in DMF for 20 min. The resin is filtered and washed with DMF (3x). The 4- methylpiperidine treatment is repeated once. The resin is subsequently filtered and washed with

DMF (3x), MeOH (3x), CH2Cl2 (3x) and DMF (3x).

288

Part 3: Experimental Procedures

XIII.9.2 Nosyl coupling

O O O O NO2 5 eq o-Ns-Cl, 10 eq collidine, S O NH2 O N CH2Cl2, r.t, 2 x 1 h H OAc OAc

VIII.19 VIII.20

To a suspension of resin VIII.19 (0.1 g, 0.0484 mmol, 1 eq) in dry DCE (1.2 ml) is added 2,4,6- collidine (64 μl, 0.464 mmol, 10 eq) and 2-nitrobenzenesulfonyl chloride (54 mg, 0.2418 mmol, 5 eq). After 1 hour shaking, the resin is filtered and washed with DMF (3x10 ml), MeOH (3x10 ml) and CH2Cl2 (3x10 ml). This procedure is repeated once, delivering the nosyl protected resin bound amino acid VIII.20.

XIII.9.3 Mitsunobu-Fukuyama alkylation

O NO O O O NO2 O O 2 S S O N O N 10 eq MeOH, 5 eq Ph3P, 5 eq DIAD, H DCE, r.t, 3x2h OAc OAc

VIII.20 VIII.21

The resin was suspended in DCE (1.2 ml) and triphenylphosphine (64 mg, 0.242 mmol, 5 eq), MeOH (0.484 mmol, 10 eq) and DIAD (48 μl, 0.242 mmol, 5 eq) were sequentially added. The reaction mixture is shaken for 2 h at room temperature after which the resin is drained and washed with dry dichloromethane and dry DCE. This Mitsunobu procedure was repeated twice, the resin was filtered and washed with DMF (3x10 ml), MeOH (3x10 ml) and CH2Cl2 (3x10 ml).

289

Part 3: Experimental Procedures

Molecular Formula: C22H22N2O6S O O O NO2 S HO N Molecular Weight: 442.48 g mol-1. Me LCMS Peak at 5.1 min, ES-MS negative mode [m/z (fragment, intensity)]: 441.0 (M-H+, 100).

XIII.9.4 Nosyl removal

O O O NO2 O S O N 2.5 eq DBU, 5 eq HSCH2CH2OH O NH DMF, r.t, 2x30min OAc OAc

VIII.21 VIII.22

The resin is suspended in DMF (1 ml) and DBU (18 μl, 0.121 mmol, 2.5 eq) and 2-mercaptoethanol (17 μl, 0.242 mmol, 5 eq) were added. The resulting suspension was shaken for 30 min after which the resin was drained and washed with DMF (3x10 ml), MeOH (3x10 ml) and CH2Cl2 (3x10 ml). This procedure is repeated once, after which the resin was drained and washed with DMF (3x10ml).

XIII.9.5 Coupling of Fmoc-anthranilic acid

O O O NHFmoc

O NH O N 10 eq anthranilic acid, 5 eq DIC OAc CH2Cl2/DMF 9/1, r.t, 24 h OAc

VIII.22 VIII.23

290

Part 3: Experimental Procedures

Preactivation: To a cooled solution (0°C) of the appropriate N-Fmoc-anthranilic acid (0.484 mmol, 10 eq) in CH2Cl2/DMF (9/1) (1.2 ml), is added DIC (26 μl, 0.242 mmol, 5 eq) and the reaction mixture was stirred for 30 min at 0 °C.

Coupling: The crude preactivation mixture was transferred to the appropriate resin VIII.22, preswollen in CH2Cl2/DMF 9/1, and the resulting suspension is shaken for 24 h. The resin was drained and washed consecutively with DMF (3x10 ml), MeOH (3x10 ml) and CH2Cl2 (3x10 ml).

XIII.9.6 Fmoc removal

O O NHFmoc O O NH2

O N 20 % 4-methylpiperidine in DMF O N

OAc r.t, 2x20min OAc

VIII.23 VIII.24

The resin is washed with DMF (3x) and treated with a solution of 20% 4-methylpiperidine in DMF for 20 min. The resin is filtered and washed with DMF (3x). The 4-methylpiperidine treatment is repeated once. The resin is subsequently filtered and washed with DMF (3x), MeOH (3x), CH2Cl2 (3x) and DMF (3x).

XIII.9.7 Cleavage from the resin

O O NH2 O O NH2 O N HO N TFA/H2O 9/1, 1 h. OAc

VIII.24 VIII.25

291

Part 3: Experimental Procedures

Resin VIII.24 is suspended in a mixture of trifluoroacetic acid/water 95/5 (5 ml) and shaken for 1 hour at room temperature. The resin is filtered and washed extensively with dichloromethane. The filtrate is concentrated under reduced pressure and the obtained residue is further purified by flash chromatography (F.C.) in dichloromethane/MeOH 99/1 to 95/5.

XIII.9.7.1 3-(N-ANTHRANOYL,N-METHYL)-2-METHYL-2-(NAPHTH-2- YLMETHYL)PROPIONIC ACID VIII.25

SPS yield 94% (white foam) O O NH2 Molecular Formula: C23H24N2O3 HO N Me Molecular Weight: 376.45 g mol-1.

LCMS Peak at 4.6 min, ES-MS positive mode [m/z (fragment, intensity)]: 377.2 (M+H+, 100).

1 H NMR: (300 MHz, CDCl3): δ 7.85-7.61 (m, 4H), 7.50-7.38 (m, 2H), 7.29 (br.s, 1H), 7.25-7.01 (m, 2H), 6.77-6.58 (m, 2H), 5.86 (br.s, 2H), 4.05-3.82 (m, 2H), 3.51-3.37 (m, 1H), 2.99 (s, 3H), 2.95-2.77 (m, 1H) 1.22 (br.s, 3H) ppm.

13 C NMR: (75 MHz, CDCl3): δ 133.3 (C), 132.3 (C), 130.7 (CH), 129.1 (CH), 128.6 (CH), 127.9 (CH), 127.7 (CH), 127.7 (CH), 127.5 (CH), 125.9 (CH), 125.6 (CH), 117.7 (CH), 116.6 (CH), 56.5

(CH2), 51.5 (CH3), 48.6 (CH2), 37.5 (CH3) ppm.

TLC: Rf = 0.3 (DCM/MeOH 9/1).

XIII.9.8 Cyclization in solution

O O O NH2 H N

HO N DCC-PS, CH2Cl2, Me 0.01M, 1h, r.t N O

VIII.25 VIII.26

292

Part 3: Experimental Procedures

To a solution of purified α,ω-aminocarboxylic acid (0.048 mmol, 1eq) in CH2Cl2 (4.8 ml) is added polystyrene-bound DCC (loading 2.3 mmol/g, 0.06 mmol, 2.9 eq). The obtained suspension is shaken for 1 h at room temperature after which the resin was filtered and washed with CH2Cl2. The filtrate was concentrated under reduced pressure. The obtained crude compound is purified by flash chromatography (F.C.) in dichloromethane/methanol 99/1.

XIII.9.8.1 3,4-DIHYDRO-3,5-DIMETHYL-3-(NAPHTH-2- YLMETHYL)BENZO[g][1,5]DIAZOCINE-2(1H),6(5H)-DIONE VIII.26

CYC yield: 60% H O N Molecular Formula: C23H22N2O2

N Molecular Weight: 358.43 g mol-1. O LCMS Peak at 5.8 min, ES-MS positive mode [m/z (fragment, intensity)]: 717.2 (2M+H+, 100), 359.2 (M+H+, 65).

1 H NMR: (300 MHz, Acetone-d6): presence of two conformers (ratio 86/14, individual assignment not possible): 8.53 (br.s, NH), 7.92-7.74 (m, 3H), 7.63 (br.s, 1H), 7.51-7.43 (m, 2H), 7.44-7.23 (m, 4H), 7.20-7.12 (m, 1H), 3.90 (d, J=15.7 Hz, 1H), 3.22 (d, J=15.7 Hz, 1H), 3.20 (s, 0.42 H), 3.16 (s, 2.58 H), 2.93 (d, J=13.4 Hz, 1H), 2.76 (d, J=13.4 Hz, 1H), 1.37 (s, 3H) ppm.

13 C NMR: (300 MHz, Acetone-d6): no multiple conformations observed: δ 174.7 (CO), 169.8 (CO), 136.1 (C), 135.8 (C), 135.1 (C), 134.3 (C), 133.4 (C), 131.2 (CH), 129.6 (CH), 129.4 (CH), 128.7 (CH), 128.4 (CH), 128.4 (CH), 128.3 (CH), 127.9 (CH), 127.1 (CH), 126.8 (CH), 126.4

(CH), 60.7 (CH2), 50.8 (C), 44.6 (CH2), 35.9 (CH3), 25.8 (CH3), ppm.

TLC: Rf = 0.25 (DCM/MeOH 9/1).

293